High Yield Functional Expression, Purification, and Reconstitution of Human Sodium/D- Glucose Cotransporter 1 (hSGLT1) in Pichia pastoris & Pilot Studies for Photoaffinity Labeling of Functional Domains of SGLT1 Dissertation Submitted to the Department of Chemistry University of Dortmund, Germany For the Degree of Doctor of Natural Sciences Completed at Max Planck Institute for Molecular Physiology, Dortmund, Germany By Navneet Kumar Tyagi Dortmund 2005 The research work described in this thesis was carried out in the period from November 2001 to November 2004 at the Max Planck Institute for Molecular Physiology, Dortmund, Germany, under the supervision of Prof. Dr. Dr. h.c. Rolf K.H. Kinne in the department of Epithelial Cell Physiology. 1. Referee: Prof. Dr. Dr. h.c. Rolf K.H. Kinne 2. Referee: Prof. Dr. Herbert Waldmann “This thesis work is dedicated to My wonderful, awesome and precious parents Siyawati and Vijai Who have demonstrated the true meaning of the word `Love` By their endless sacrifices, bountiful care, and unshakable determination for my well being throughout every day of their lives”. “ The beauty and frustration of science is that it constantly produces surprises” “ Unknown” Table of Contents i Abbreviations.................................................................................................................. ............v 1. Introduction ............................................................................................................................ 1 1.1. Transporters..................................................................................................................... 1 1.2. Solute carrier (SLC) families of transporters .................................................................. 2 1.2.1. Sodium/glucose cotransporter family (SLC5).......................................................... 3 1.2.2. Sodium/glucose cotransporter protein (SGLT1) ...................................................... 8 1.3. Pichia pastoris as an expression host............................................................................ 10 1.4. Photoaffinity labeling .................................................................................................... 11 2. Aim of the study ............................................................................................................ ....... 13 3. Materials and methods....................................................................................................... ... 14 3.1. General equipments....................................................................................................... 1 4 3.2. Materials................................................................................................................. ....... 15 3.2.1. Chemicals ............................................................................................................... 15 3.2.2. Enzymes and reagents for molecular biology ........................................................ 16 3.2.3. Antibodies .............................................................................................................. 16 3.2.4. Commercial Kits and other materials ..................................................................... 16 3.2.5. Primers for preparation of cDNA inserts ............................................................... 17 3.2.6. Plasmids................................................................................................................ .. 17 3.2.6.1. Original plasmids ............................................................................................ 17 3.2.6.2. Plasmid with insert .......................................................................................... 18 3.3. Work with E.coli ........................................................................................................... 19 3.3.1. E.coli strains ........................................................................................................... 19 3.3.2. Media for bacterial culture ..................................................................................... 19 3.3.3. General ................................................................................................................. .. 20 3.3.4. Transformation of plasmid in E.coli....................................................................... 20 3.3.4.1. Preparation of competent cells by CaCl 2 method............................................ 20 3.3.4.2. Heat shock transformation of the plasmid....................................................... 20 3.4. Work with P. pastoris ................................................................................................... 20 3.4.1. P. pastoris strains ................................................................................................... 20 3.4.2. Media for P. pastoris culture.................................................................................. 21 3.4.3. General ................................................................................................................. .. 21 3.4.4. Electroporation of P. pastoris with plasmid pPICZB or pPICZB-hSGLT1 .......... 22 3.5. Recombinant DNA processing and manipulation ......................................................... 22 3.5.1. Buffers and solutions.............................................................................................. 22 3.5.2. Restriction endonuclease digestion of DNA .......................................................... 23 3.5.3. Purification of the digested DNA ........................................................................... 23 3.5.4. Ethanol precipitation of DNA ................................................................................ 23 3.5.5. Dephosphorylation of lin earized plasmid DNA by CIP......................................... 23 3.5.6. Ligation of DNA fragments ................................................................................... 24 Table of Contents ii 3.5.7. Miniprep: small-scale preparation of plasmid DNA .............................................. 24 3.5.8. Large scale preparation of plasmid DNA............................................................... 24 3.5.9. Qunatification of DNA and RNA solutions ........................................................... 25 3.5.10. Agrose gel electrophoresis ................................................................................... 25 3.5.11. DNA recovery from agarose gel .......................................................................... 26 3.5.12. DNA sequencing .................................................................................................. 26 3.5.13. DNA amplification of hSGLT1 gene by PCR for cloning into different expression systems ............................................................................................................................. 26 3.5.14. Addition or deletion mutagenesis in hSGLT1 gene ............................................. 27 3.5.14.1. Addition of 30 base pairs long spacer at the C-terminal of hSGLT1 gene ... 29 3.5.14.2. Addition of FLAG tag in hSGLT-spacer gene.............................................. 29 3.5.14.3. Deletion of N-terminal half of hSGLT1 gene ............................................... 29 3.5.15. Addition of Unc-F gene at the N-terminal of hSGLT1 gene ............................... 30 3.6. Protein expression and analysis..................................................................................... 30 3.6.1. Growth conditions of E.coli for hSGLT1 expression............................................. 30 3.6.2. Growth condition of P. pastoris for hSGLT1 expression ...................................... 31 3.6.3. hSGLT1 purification from P. pastoris ................................................................... 31 3.7. Protein analysis.......................................................................................................... .... 32 3.7.1. Buffers and solutions.............................................................................................. 32 3.7.2. Measurement of protein concentration................................................................... 33 3.7.2.1. Enhanced alkaline copper (Lowry) protein assay ........................................... 33 3.7.3. SDS-PAGE............................................................................................................. 33 3.7.4. Coomasie staining of the polyacrylamide gels....................................................... 34 3.7.5. Western blot analysis.............................................................................................. 34 3.8. Functional analysis of recombinant hSGLT1................................................................ 35 3.8.1. Membrane preparations for transport studies......................................................... 35 3.8.2. Reconstitution of recombinant hSGLT1 ................................................................ 36 3.8.3. Transport assay of hSGLT1 in membranes and in proteoliposomes...................... 36 3.9. Chemical synthesis of photoaffinity probes .................................................................. 37 3.9.1. [(2’-Iodo -4’- ( 3 ’ ’ -trifluoromethyldiazirinyl) phenoxy]-D -glucopyranoside (TIPDG) .......................................................................................................................................... 37 3.9.1.1. 4-Methoxytrifluoroacetophenone (1) .............................................................. 37 3.9.1.2. 3-Iodo-4-methoxytrifluoroacetophenone (2)................................................... 37 3.9.1.3. 4-Hydroxy-3-iodo-tr ifluoroacetophenone (3) ................................................. 37 3.9.1.4. [(2´-Iodo-4´-trifluoroacetyl ) phenoxy]-2,3,4,6-tetra-O-acetyl-D- glucopyranoside (5) ...................................................................................................... 37 3.9.1.5. [(2´-Iodo-4´-trifluoroacetyloxi me) phenoxy]-2,3,4,6-tetra-O-acetyl-D- glucopyranoside (6) ...................................................................................................... 38 Table of Contents iii 3.9.1.6. [(2´-Iodo-4´-(3´´-trifluoromethyldi aziridinyl) phenoxy]-2,3,4,6-tetra-O-acetyl- D- glucopyranoside (7)................................................................................................. 38 3.9.1.7. [(2´-Iodo-4´-(3´´-trifluoromethyl diazirinyl) phenoxy]-2,3,4,6-tetra-O-acetyl- D- glucopyranoside (8)................................................................................................. 39 3.9.1.8. [(2´-Iodo-4´-(3´´-trifluoromethyl diazirinyl) phenoxy]-D-glucopyranoside (9) ...................................................................................................................................... 40 3.9.2. [(4´-Benzoyl) phenoxy] -D- glucopyranoside (BzG) .............................................. 40 3.9.2.1. [4´-Benzoyl) phenoxy] –2,3,4,6-tetra-O-acetyl-D- glucopyranoside (11) ...... 40 3.9.2.2. [(4´-Benzoyl) phenoxy]-D- glucopyranoside (12) .......................................... 40 3.9.3. 3-Azidophlorizin (3-AP) ........................................................................................ 41 3.9.3.1. 3-Nitrophlorizin (14) ....................................................................................... 41 3.9.3.2. 3-Aminophorizin (15)...................................................................................... 41 3.9.3.3. 3-Azidophlorizin (16) ...................................................................................... 42 3.9.4. n-Methyl-6C- (Azimet hyl)-D- glucopyranoside (6-AG) ....................................... 42 3.9.4.1. Methyl-2,3,4-tri-O-benzyl-7-de oxy-DL-glycero-D-glucohepta-1,5- pyranoside (18) ........................................................................................................................... .... 42 3.9.4.2. Methyl-2,3,4-tri-O-benzyl-7-de oxy-D-glucohepto-1,5-pyranoside-6-ulose (19) ...................................................................................................................................... 43 3.9.4.3. Methyl- 7- deoxy-D-glucohepta- 1, 5- pyranoside-6- ulose (20) .................... 43 3.9.4.4. n-Methyl-6C-(Azimethyl)-D-glucopyranoside (22)........................................ 44 3.10. Photoaffinity labeling of loop 13................................................................................. 45 3.10.1. Isolation of small intestine br ush border membrane vesicles (BBMV) ............... 45 3.10.2. Transport measurements....................................................................................... 45 3.10.3. Photoaffinity labeling of truncated loop 13 protein with TIPDG or BzG............ 45 3.10.4. Photoaffinity labeling of trun cated loop 13 protein with 3-AP............................ 46 4. Results .................................................................................................................................. 48 4.1. E.coli as an expression host........................................................................................... 48 4.1.1. Construction of different clones of hSGLT1 in pET22b vector............................. 48 4.1.2. Construction of pET22b-Unc-F and pET22b-Unc-F-hSGLT1 clones................... 49 4.1.3. hSGLT1 expression in E.coli ................................................................................. 50 4.1.4. β -subunit of E.coli ATPase expression in pET22b-Unc-F and pET22b-Unc-F- hSGLT1 plasmids............................................................................................................. 51 4.1.5. Messenger RNA stability of hSGLT1 in E.coli ..................................................... 52 4.2. Pichia pastoris as an expression host............................................................................ 54 4.2.1. Construction of expression plasmid pPICZB-hSGLT1.......................................... 54 4.2.2. Identification of positive clones of hSGLT1 showing protein expression............. 55 4.2.3. Optimization of feeding and in duction in the shake flask cultures ........................ 55 4.2.4. Purification of hSGLT1 from Pichia pastoris........................................................ 56 Table of Contents iv 4.2.5. Functional analysis of recombinant hSGLT1 in right-side-out membrane vesicles .......................................................................................................................................... 57 4.2.6. Functional analysis of recombinant hSGLT1 in prteoliposomes ........................... 58 4.3. Synthesis and Characterization of different photoaffinity probes................................. 63 4.3.1. Synthesis of labels BzG and 3-AP for the identification of phlorizin interaction sites................................................................................................................................... 63 4.3.2. Synthesis of label TIPDG for the id entification of arbutin ineraction sites ........... 64 4.3.3. Synthesis of label 6-AG for the identi fication of D-glucose interaction sites........ 64 4.3.4. Photochemical properties and stabil ity of TIPDG, BzG, 3-AP and 6-AG............. 65 4.3.5. Inhibition of glucose uptake in rabbi t small intestine BBMV by BzG and 3-AP .. 66 4.3.6. Inhibition of glucose uptake in rabbit small intestine BBMV by TIPDG.............. 67 4.3.7. Inhibition of glucose uptake in rabbit small intestine BBMV by 6-AG ................ 68 4.4. Photoaffinity labeling of truncated loop 13 with TIPDG, BzG, and 3-AP ................... 70 4.4.1. Photoaffinity labeling of truncated loop 13 with TIPDG and BzG........................ 70 4.4.2. Photoaffinity labeling of truncated loop 13 with 3- Azidophlorizin ...................... 71 4.4.3. Identification of th e ligand contact points .............................................................. 72 5. Discussion ............................................................................................................................ 74 5.1. E. coli as an expression host.......................................................................................... 75 5.1.1. Probable reasons for not getting hSGLT1 expression in E. coli ............................ 75 5.1.1.1. Codon usage .................................................................................................... 75 5.1.1.2. Signal sequences.............................................................................................. 76 5.1.1.3. Degradation of hSGLT1 mRNA in E.coli....................................................... 76 5.1.1.4. Comparison to other attempts to express hSGLT1 in E. coli .......................... 78 5.2. P. pastoris as an expression host................................................................................... 80 5.2.1. Expression of hSGLT1 in P. pastoris .................................................................... 80 5.2.2. Protein purification................................................................................................. 81 5.2.3. Reconstitution and functional an alysis of recombinant hSGLT1 .......................... 82 5.2.4. Reasons for getting functional expression of hSGLT1 in P. pastoris.................... 82 5.2.5. Advantages of our expression method ................................................................... 83 5.3. Photoaffinity labeling of truncated loop 13................................................................... 84 5.4. Future prospective ......................................................................................................... 87 6. Summary ..................................................................................................................... ......... 89 7. Zusammenfassung ............................................................................................................. ... 91 8. Reference list .............................................................................................................. .......... 93 Erklärung Acknowledgements Curriculum Vitae Abbreviations v Abbreviations aa Amino Acids Ac Acetate 6-AG n-Methyl-6C- (azimethyl)-D- glucopyranoside Amp Ampicillin 3-AP 3- Azidophlorizin Arg Arginine ATP (ADP, AMP) Adenosine-5´-tri (Di, Mono-) Phosphate BBMV Brush border membrane vesicles bp Base Pair BSA Bovine Serum Albumin BzG [(4´-Benzoyl)phenoxy]-D- glucopyranoside cDNA Complimentary DNA CIAP Calf Intestine Alkaline Phosphatase DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid dNTP Deoxy-Nucleotriphosphate DTT Dithiothreitol EDTA Ethylenediaminetetraacetate EST Expressed Sequence Tag EtBr Ethidium bromide g Gram h Hours hSGLT1 Human sodium/D- glucose cotransporter 1 IPTG Isopropyl- β -D-Thioglucoside Ki Inhibition constant M Molar MALDI-TOF Matrix-assisted laser desorption ionization –time of flight MCS Multiple Cloning Site Min Minute ml Milliliter mM Milli Molar NC Nitro Cellulose membrane OD Optical Density PAGE Polyacrylamide Gel Electrophoresis PCR Polymerase Chain Reaction RNA Ribonucleic acid RT Room Temperature Abbreviations vi SDS Sodium Dodecylsulphate TCA Trichloroacetic acid TEMED N, N, N´, N´ Tetramethylethylenediamine TIPDG [(2’-Iodo -4’- ( 3 ’ ’ -trifluoromethyldiazirinyl)phenoxy]-D - glucopyranoside TMH Trans Membrane Helices Tris Tris (hydroxymethy) amino methane Trp Tryptophan U Unit UV Ultraviolet Introduction 1 1. Introduction 1.1. Transporters Transporters are the gatekeepers for all cells and organelles, facilitating influx and efflux of crucial compounds such as sugars, amino acids, nucleotides, inorganic ions and drugs. Transporters can be divided into passive and active transporters (Figure 1.1). Passive transporters, allow passage of solutes (e.g., glucose, amino acid, urea) across membranes down their electrochemical gradients. Active tr ansporters create ion/solute gradients across membranes, utilizing diverse energy-coupling mechanisms. These active transporters are classified as primary- or secondary-active tran sporters according to the directness of coupling to cellular energy (e.g., ATP hydrolysis, or ion gradients). Figure 1.1 Cartoon showing a cell with different transporters. Solute carrier (SLC)- and non-SLC transporters expressed in the plasma membrane or in intracellular compartment membranes are shown. Primary-active, ATP-dependent transporters include members of the ATP-binding cassette (ABC) transporter family and ion pumps (ATPases). Mammalian ABC transporters [e.g., P- glycoprotein/multi-drug resistance (MDR) proteins and transporter associated with antigen processing (TAP)] bind or hydroly ze ATP as a control gate for the transport of a variety of substances such as ions, carbohydrates, lipids, xenobiotics and drugs, out of cells or into organelles (Borst and Elferink, 2002). Ion pum ps hydrolyze ATP to pump ions such as Na + , K+ , H+ , Ca2+ and Cu2+ out of cells or into organelles, for review see (Dunbar and Caplan, Introduction 2 2000; Muller and Gruber, 2003). These pumps also generate and maintain electrochemical ion gradients across membrane, and are thus called active transporters. Such ion gradients are used in turn by secondary-active, ion-coupled tr ansporters to drive uphill transport of nutrients across biological membranes. Similar to transporter, channels allow movement of solute down their electrochemical gradients, for review see (Armstrong, 2003; Chen, 2003; Decoursey, 2003; Jiang et al., 2003; Yu and Catterall, 2003). Transporters typically have a fixed stoichiometry of ion/solute movement per translocation cycle. Ion or solute flow through channels, on the other hand, is controlled by open probability of the channel via gating mechanisms. 1.2. Solute carrier (SLC) families of transporters The solute carrier (SLC) series includes gene s encoding passive transporters, ion compled transporters and exchangers (Hediger et al ., 2004; Wright and Turk, 2004). A transporter has been assigned to a specific SLC family if it has at least 20-25% amino acid sequence identity to other members of that family (Turk and Wright, 1997). The list of currently approved SLC human gene symbols is shown in Table 1.1. The table comprises 43 different SLC transporter families of the SLC series and the number of members in each family. Table 1.1 List of currently approve d solute carrier (SLC) families. The HUGO SLC Family Series Total The high-affinity glutamate and neutral amino acid transporter family 7 SLC1 SLC2 The facilitative GLUT transporter family 14 SLC3 The heavy subunits of the heteromeric amino acid transporters 2 SLC4 The bicarbonate transporter family 10 SLC5 The Na + /glucose cotransporter family 8 SLC6 The sodium- and chloride-dependent neurotransmitter transporter family 16 SLC7 The cationic amino acid transporter/glycoprotein-associated amino-acid transporter family 14 SLC8 The Na + /Ca 2+ exchanger family 3 SLC9 The Na + /H + exchanger family 8 SLC10 The sodium bile salt cotransport family 6 SLC11 The proton-coupled metal ion transporter family 2 SLC12 The electroneutral cation-Cl cotransporter family 9 SLC13 The human Na + -sulfate/carboxylate cotransporter family 5 SLC14 The urea transporter family 2 SLC15 The proton oligopeptide cotransporter family 4 SLC16 The monocarboxylate transporter family 14 SLC17 The vesicular glutamate transporter family 8 SLC18 The vesicular amine transporter family 3 SLC19 The folate/thiamine transporter family 3 SLC20 The type-III Na + -phosphate cotransporter family 2 Introduction 3 SLC21/SLCO The organic anion transporting family 11 SLC22 The organic cation/anion/zwitterion transporter family 18 SLC23 The Na + -dependent ascorbic acid transporter family 4 SLC24 The Na + /(Ca 2+ -K + ) exchanger family 5 SLC25 The mitochondrial carrier family 27 SLC26 The multifunctional anion exchanger family 10 SLC27 The fatty acid transport protein family 6 SLC28 The Na + -coupled nucleoside transport family 3 SLC29 The facilitative nucleoside transporter family 4 SLC30 The zinc efflux family 9 SLC31 The copper transporter family 2 SLC32 The vesicular inhibitory amino acid transporter family 1 SLC33 The acetyl-CoA transporter family 1 SLC34 The type-II Na + -phosphate cotransporter family 3 SLC35 The nucleoside-sugar transporter family 17 SLC36 The proton-coupled amino acid transporter family 4 SLC37 The sugar-phosphate/phosphate exchanger family 4 SLC38 The System A and N, s odium-coupled neutral amino acid transporter family 6 SLC39 The metal ion transporter family 14 SLC40 The basolateral iron transporter family 1 SLC41 The MgtE-like magnesium transporter family 3 SLC42 The Rh ammonium transporter family (pending) 3 SLC 43 The Na + -independent, system- L -like amino acid transporter family 2 Total 298 In general the genes are named using the root symbol SLC, followed by a numeral (e.g., SLC1, solute carrier family 1), the letter A (which act as a divider between the numerals) and finally the number of the individual transporter (e.g., SLC5A1, sodium/glucose cotransporter protein). These general rules of SLC gene nomen clature have been elaborated further for a couple of families. In the nucleotide-sugar tr ansporter family SLC35, the letter between SLC35 and family member number has been exploited to specify specific subfamilies, called A, B, C, D and E (Ishida and Kawakita, 2004). In another family, originally named SLC21, encoding the organic anion-transporting (OATP) proteins, the “21” and the “A” have been replaced by the letter “O”, which stands for organic transporter. This modification has occurred to accommodate a unique, species-independe nt classification and naming system that has been introduced, because this family has been the subject of rapid evolution, giving rise to new isoforms within a given species (Hagenbuch and Meier, 2004). 1.2.1. Sodium/glucose cotransporter family (SLC5) The sodium/glucose cotransporter family (SLC5) has 220 members in animal and bacterial cells (Wright and Turk, 2004). An unrooted phylogenetic tree of 11 human members of the SLC5 family of cotransporters and two other vertebrate members of known function is shown Introduction 4 in Figure 1.2. The 11 human genes (Table 1.2) are expressed in tissues such as the small intestine, kidney, brain, muscle, and thyroid gland. Despite the homology among the proteins (21-70% amino acid identity to SGLT1) there is di versity in gene structure: in eight of ten genes mapped the coding sequences are found in 14-15 exons (SGLT1-6, NIS, and the Na + - dependent multivitamin transporter SMVT) (T urk et al., 1994; Wang et al., 1999). On the other hand the coding sequences for the choline transporter (CHT) and SMIT are contained in eight and one exons respectively (Mallee et al., 1997; Okuda et al., 2000). In SGLT4-6 and SMIT there is evidence for alternative splicing (Roll et al., 2002). Figure 1.2 Unrooted phylogenetic tree of the 11 human members of the SLC5 family of cotransporters and two other vertebrate members of known function. Members for which transport functions have been demonstrated experimentally are shown in red. Members shown in black have yet to have their function determined. In a pairwise basic local alignment search tool (BLAST) analysis the amino acid identities relative to SGLT1 were 59% SGLT2, 70% SGLT3, 56% SGLT4, 57% SGLT5, 50% SGLT6, 55% SMIT, 24% SMVT, 24% CHT, and 21% NIS ( SGLT Na+ /glucose transporter, SMIT Na+ / myo -inositol transporter, SMVT Na+ /multivitamin transporter, CHT choline transporter, AIT apical iodide transporter, NIS Na+ /iodide transporter). Int ro du ct io n 5 T ab le 1 .2 T he S L C 5 so di um /g lu co se co tr an sp or te r fa m ily (OMIM On lin e Men de lia n Inh er ita nc e in Ma n D at ab as e, TMH tr an sm em br an e he lix , a a a m in o ac id s) . H um an ge ne n am e Pr ot ei n na m e Alia se s Pr ed om in an t su bs tr at es T ra ns po rt ty pe /c ou pl in g io n* T is su es di st ri bu tio n an d ce llu la r/ su bc el lu la r ex pr es si on T L in k to d is ea se H um an ge ne lo cu Se que nc e Acc es si on ID Sp lic e va ri an ts an d th ei r sp ec ifi c fe at ur es S SLC 5A1 SG LT1 N on e G lu co se an d ga la ct os e (ur ea an d w at er ) C /N a + (H + ) F/N a + (H + ). C ha nn el : ur ea an d w at er Sm al l in te st in e>>t ra ch ea , ki dn ey an d he ar t; pl as m a m em br an es G lu co se ga la ct os e m al ab so rp tio nG OM IM 1 82380 22 q13. 1 N M _00 034 3 SLC 5A2 SG LT2 N on e G lu co se C /N a + K id ne y co rte x Fa m ili al re na l gl yc os ur ia G OM IM 1 82381 16p 12 - p1 1 N M _00 304 1 SLC 5A3 SM IT N on e m yo -in os ito l (gl uc os e) C /N a + B ra in , h ea rt, k id ne y an d lu ng ; pl as m a m em br an es D ow n’s sy nd ro m e? OM IM 60 04 44 21 q22 .1 2 N M _00 6933 SLC 5A4 SG LT3 SAAT 1 N a+ (H + ) G lu co se ac tiv at ed N a+ (H + ) c ha nn el Sm al l in te st in e (ch ol in er gi c ne ur on s), sk el et al m us cl e, ki dn ey , ut er us an d te st is ; pl as m a m em br an es 22 q12 .2 - q12 .3 N M _14 22 7 SLC 5A5 N IS I– (C lO 4 – , SC N – , N O 3 – , B r– ) C /N a + U ni po rte r N a+ ch an ne l U re a w at er Th yr oi d, br ea st , co lo n an d ov ar y; pl as m a m em br an es Th yr oi d ho rm on og en es is G OM IM 60 184 3 19p 13. 2- p1 2 N M _ 0 00 45 3 SLC 5A6 SM VT B io tin , lip oa te an d pa nt ot he na te C /N a + B ra in , h ea rt, k id ne y, lu ng an d pl ac en ta ; pl as m a m em br an es 2p 23 N M _0 21 095 SLC 5A7 C H T C H T1 C ho lin e C /N a + /C l– Sp in al co rd an d m ed ul la 2q1 2 N M _0 21 815 Int ro du ct io n 6 (in tra ce llu la r ve si cl es ) SLC 5A8** SG LT4 Sm al l in te st in e, ki dn ey , liv er , lu ng an d br ai n 1p 32 H C T1 951 464 In te rn al sp lic e in Ex on 14 ad ds ei th er 38 or 53 aa be tw ee n TM H s1 3- 14 SLC 5A9 SG LT5 R ab bi t R K -D K id ne y 17p 11 .2 XM _064 487 Ex on 7 m ay be sp lic ed ou t de le tin g 26 aa be tw ee n TM H s 5– 6; in te rn al sp lic e in ex on 10 m ea ns ei th er 36 or 52 aa be tw ee n TM H s 8– 9; a nd a n in te rn al sp lic e in ex on 12 ad ds Int ro du ct io n 7 ei th er 12 or 37 aa be tw ee n TM H s 11 –12 SLC 5A1 0 SG LT6 K ST 1 R ab bi t ST 1 R ab bi t SM IT 2 R ab bi t or th ol og : m yo -in os ito l, ch iro - in os ito l, gl uc os e an d xy lo se . Xen op us la ev is or th ol og : m yo -in os ito l an d gl uc os e C /N a + Sm al l in te st in e, br ai n, k id ne y, l iv er , he ar t, an d lu ng 16p 12 .1 N M _05 294 4 Sp lic in g el im in at es ex on 6 an d TM H 4 SLC 5A1 1 AIT Io di de Th yr oi d; ap ic al pl as m a m em br an e 12 q23. 1 N M _14 591 3 *C c ot ra ns po rte r; F u ni po rte r. Fu nc tio n ba se d on re su lts o bt ai ne d w ith h et er ol og ou s e xp re ss io n sy st em s ** P ro vi si on al S G LT4 e xo ns id en tif ie d by m in in g th e C el er a da ta ba se s T Ti ss ue d is tri bu tio n of S G LT1 -6 w as d et er m in ed R N Aas e pr ot ec tio n as sa ys u si ng g en e sp ec ifi c pr ob es . Al so i nc lu de s da ta f ro m t he o rig in al cl on in g pa pe rs a nd G en eC ar d (ES T, a nd /or D N A a rr ay ) G G en e de fe ct S P ot en tia l a lte rn at iv e sp lic e si te s N ot e: si ng le n uc le ot id e po ly m or ph is m s (S N Ps ) a nd v ar ia n ts in th e N C B I S N P da ta ba se a re n ot in cl ud ed . Introduction 8 1.2.2. Sodium/glucose cotransporter protein (SGLT1) SGLT1 (SLC5A1) was expression-cloned in 1987 (Hediger et al., 1987). SGLT1 is expressed primarily in the brush border membrane of mature enterocytes in the small intestine where it plays a central role for the homeostasis of glucose, galactose, salt and water (Kinne, 1976; Wright et al., 1994; Wright a nd Loo, 2000; Wright et al., 2001). The most well known genetic disease caused by SGLT1 is glucose-galactose malabsorption (GGM) (Kasahara et al., 2001; Lam et al., 1999; Martin et al., 1997; Martin et al., 1996; Turk et al ., 1991; Wright et al., 2001; Wright et al., 2002). This is a rare autosomal recessive disease (Online Mendelian Inheritance in Man database accession No. OMIM 182380) that presents in newborn infants as a life-threatening diarrhea. The diarrhea ceases promptly on removing dietary glucose, galactose and lactose, but returns immediately on reintroducing one of the offending sugars into the child’s diet. In some 46 patients tested sofar mutations have been identified in the SGLT1 gene that cause the defect in sugar transport. These include missense, nonsense, frame-shift, and splice-site mutations. The misse nse mutations were studied using the oocytes expression system, where the defect in transport in 22 out of 23 mutants was due to missorting of the protein in the cell. Surprisingly, some very conservative GGM mutations cause the trafficking defect; e.g., four are alanine to va line trafficking mutants. Normal trafficking is restored when valine is replaced by cysteine, suggesting that slight conformational changes in the protein interfere with interactions between the transport vesicles containing SGLT1 and motor proteins responsible for delivery of the vesicle to plasma membrane. Human isoforms of SGLT1 are 664 ami no acids long with 14 transmembrane α-helices and both the N- and C-terminals facing the extrace llular fluid (Turk and Wright, 1997) (Figure 1.3). A secondary structure model as shown in Figure 1.3 is indicati ng position of loop 13 as intracellular (Turk and Wright, 1997), while our group (Lin et al., 1999) results with 6 × Histidine tagged mutant in COS-7 cells indicates that the last large group (loop13) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition. However both the secondary structure models are not able to explain all the functional properties of SGLT1, so both models are not 100% correct, but it might be the case that this loop exists in dynamic equilibrium between extr acellular and intracellular orientations, which takes place during the interaction with substrates. Extensive molecular biological and biophysical studies of hSGLT1 in Xenopus laevis oocytes revealed that the N-terminal half of the protein contains the Na+ -binding sites (Meinild et al., 2001) while the solutes and inhibitors binding s ites lies in the C-terminal half of protein (Panayotova-Heiermann et al., 1997; Panayotova-H eiermann et al., 1996). Further studies in this direction indicate that residue Gln-457, Thr-460 (Diez-Sampedro et al., 2001; Napoli et al., 1995) and negative charge at residue Asp-454 form the sugar-binding and translocation pathway (Diez-Sampedro et al., 2004). The mechanism by which SGLT1 couple their substrates across the cell membrane is still unknown. A commonly proposed kinetic model for Introduction 9 sodium/glucose cotransport is a six-state orde red kinetic model (Loo et al., 1993; Loo et al., 1998; Parent et al., 1992a; Parent et al ., 1992b; Peerce and Wright, 1984). The model describes sodium/glucose cotransport as a se ries of ligand-induced conformational changes (Figure 1.4). On the external surface of the membrane, two Na + ions bind to the transporter before sugar. The empty transporter C (states 1 and 6), Na + -loaded transporter CNa 2 (states 2 and 5), and the sugar-loaded transporter CNa 2S (states 3 and 4) can “cross” the membrane. Membrane voltage affects Na+ -binding and translocation of the empty transporter. Figure 1.3 Tentative secondary structure model of hSGLT1. hSGLT1 consists of 14 α-helical transmembrane domains (shaded columns) with the N- and C-terminal located on the extracellular phase of membrane. The N- glycosylation site (Asn-248; N) is indicated (Quick and Wright, 2002). Heterologous expression of hSGLT1 has been described in Xenopus laevis oocytes (Hediger et al., 1987), COS-7 cells (Birnir et al., 1990), CHO cells (Lin et al., 1998), Sf9 cells (Smith et al., 1992), and Escherichia coli ( Quick and Wright, 2002) and has increased our knowledge about functions of transporter significantly but the amount of protein produced in the above mentioned systems is not sufficient for most of the biochemical, biophysical and structural studies which need tens or some times hundreds of milligrams of highly purified protein. In view of these difficulties our group’s previous success in the expression and purification of different fragments of SGLT1 (Raja et al., 2003; Xia et al., 2003) and their subsequent use as a model for the identification of phlorizin-bindi ng site and alkyl glucosides-binding site as studied by fluorescence spectroscopy and affinity labeling (Raja et al., 2004; Raja et al., 2003; Tyagi and Kinne, 2003; Xia et al., 2003) provided valuable information about different inhibitors mechanism. However there lies an urgent need to express hSGLT1 in full-length Introduction 10 and sufficient quantity. To achieve high-level expression of hSGLT1 we chose to use the methyltropic yeast Pichia pastoris because in the last 15 years this expression system has emerged as the most successful expression system for the heterologous expression of a wide variety of foreign proteins (Cereghino and Cregg, 2000; Cregg et al., 2000; Higgins and Cregg, 1998), especially membrane proteins, wh ich were either not possible to express in E. coli or if possible, express in very low amounts often with loss of functionality. Figure 1.4 Cartoon of the 6-state ordered kinetic model for sodium/glucose cotransport. States 1-3 face the external and states 4-6 internal memb rane surfaces, respectively. In the absence of ligands, the transporter exists in 2 states (1 and 6). At the external surface, 2 Na + ions bind to the transporter to form the complex CNa2 (state 2). The fully loaded transporter CNa 2S (state 3) undergoes a conformational change (states 3 to 4) resulting in sodium/glucose cotransport. The reac tion from state 2 to 5 represents a uniport (“leak”) pathway for the sugar (Loo et al., 1998). 1.3. Pichia pastoris as an expression host Pichia pastoris has become a highly successful system for the expression of heterologous genes. Several factors have contributed to its rapid acceptance, the most important of which include: 1. A promoter derived from the alcohol oxidase I ( AOX1) gene of P. pastoris that is uniquely suited for the controlled expression of foreign genes; 2. The similarity of techniques needed for the molecular genetic manipulation of P. pastoris to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; Introduction 11 3. The strong preference of P. pastoris for respiratory growth, a key physiological trait that greatly facilitates its culturing at high cell densities relative to fermentative yeasts; and 4. A 1993 decision by Phillips Petroleum Company to release the P. pastoris expression system to academic research laboratories, the consequences of which has been an explosion in the knowledge base of the system. As a yeast, P. pastoris is a single-celled microorganism th at is easy to manipulate and to culture. However, it is also a eukaryote and capable of many of the posttranslational modifications performed by higher eukaryotic cells, such as proteolytic processing, protein folding, and disulfide bond formation. Thus many proteins that end up as inactive inclusion bodies in E. coli are produced as biologically active molecules in P. pastoris. The P. pastoris system is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from higher eukaryotes, such as insect and mammalian tissue culture cell systems, and usually gives higher expression levels. A second role played by P. pastoris in research is not directly related to its use as a protein expression system. P. pastoris serves as a useful model system to investigate certain areas of modern cell biology, including the molecular mechanisms involved in import and assembly of peroxisomes, the selective autophagic degradation of peroxisomes, and the organization and function of the secretory pathway in eukaryotes. More detailed information on the P. pastoris system can be found in the numerous reviews describing the system (Cereghino and Cregg, 2000; Cregg et al., 2000; Higgins and Cregg, 1998) and the Pichia Expression Kit Manual (Invitrogen Corporation, Carlsbad, CA, USA). The DNA sequence of many P. pastoris expression vectors and other useful information can be found on the Invitrogen web site (http:// www.invitrogen.com). In the last few years the following membrane proteins have been successfully expressed in P. pastoris, Na+ , K+ -ATPase (Strugatsky et al., 2003), multidr ug resistance protein1 (Cai et al., 2001), ATP-binding cassette transporter (Cai a nd Gros, 2003), NOP-1 (Bieszke et al., 1999), mammalian intestinal peptide transporter (Doring et al., 1998), human µ-opioid receptor (Talmont et al., 1996) and 5HT 5A -serotoninergic receptor (Weiss et al., 1998). 1.4. Photoaffinity labeling Photoaffinity labeling and cross-linking refer to a variety of important biochemical techniques that are used to investigate structural and functional properties of biological systems, for review see (Bayley, 1983; Bayley and Knowle s, 1977; Brunner, 1993; Dorman and Prestwich, 2000). These techniques make use of reagents, whic h, after targeting to a biological system or component, can be activated with UV light to generate highly reactive intermediates capable of forming covalent bonds with adjacent molecules. Labeling reagents can be divided into three main classes: first, probes designed to report on general properties of a system (for Introduction 12 example, small apolar molecules and lipids which partition into membranes and, upon activation, label selectively integral membrane proteins) (Brunner et al., 1980; Do et al., 1996; D'Silva and Lala, 2000; Heinrich et al., 2000; Mo thes et al., 1997), second, affinity labeling reagents intended to interact with and label components (receptors, cotransporters, and channels) in a specific, functionally releva nt manner (Addona et al., 2002; Jahn et al., 2002; Kipp et al., 1997; Leite et al., 2003; Thiele et al., 2000; Tyagi and Kinne, 2003), and third, heterobifunctional photo cross-linkers, cont aining a photactivatable and a conventional (thermal) reactive functional group, preferentia lly connected via a cleavable linker. These later reagents are primarily used to study the spatial relationship between components in complex systems. Several successful examples of photoaffinity labeling of membrane proteins have been reported; among these are the glucose transporters in the plasma membrane of erythrocytes and of adipocytes (Trosper and Levy, 1977), the β -galactoside transport in E. coli membrane vesicles (Rudnick et al., 1975), the dipep tide and oligopetides transporters in E. coli (Staros and Knowles, 1978), rhodopsin (Borhan et al ., 2000), the ATP-binding cassette transporter LmrA (van Veen et al., 2000), fatty acid binding proteins in E. coli (Mangroo and Gerber, 1992), alpha 1-adrenegic receptors of rat heart (Terman and Insel, 1986), anion channel-1 in rat brain (Darbandi-Tonkabon et al., 2003), the i on channel of F-ATP synthase (von Ballmoos et al., 2004), the synaptic vesicle protei n SV2A (Lynch et al., 2004), the bovine γ- aminobutyric acid type receptor (Sawyer et al., 2002), and nicotinic acetylcholine receptor (Chiara et al., 2003; Pratt et al., 2000; Wang et al., 2000; Ziebell et al., 2004). Aim 13 2. Aim of the study Large-scale purification of recombinant human membrane proteins represents a rate-limiting step towards understanding their role in health and disease. There are only four mammalian membrane proteins of known structure and these were isolated from natural sources. However, heterologous expression of hSGLT1 has been described in Xenopus laevis oocytes, COS-7, CHO, Sf9 cells, and Escherichia coli but the amount of protein produced in the above mentioned systems is not sufficient for most of the biochemical, biophysical and structural studies. In order to shed more light on the structure-functions of hSGLT we intend to express full-length transporter in high yield to fulfill our requirements of protein amount, which is necessary for most of the biochemical and biophysical experiments. The elucidation of molecular interactions leading to transport of sugar and its translocation pathway through SGLT1 is a major objective of this work. Different methods have been widely used for the identification of sugar translocation pathway into SGLT1, but all methods were unable to provide an insight into protein structure at molecular level. To overcome this problem we proposed the use of photoaffinity labeling of functional domains of SGLT1 with different ligands and inhibitors based photoaffinity probes for the identification of sugar translocation pathway and inhibitor binding sites. To achieve this aim we proposed the synthesis and application of four new photoaffinity probes. Therefore the main objectives of this work are: 1. To achieve high-level heterologous expre ssion, purification and reconstitution of full- length functional hSGLT1. 2. Functional characterization of recombinant hSGLT1 by sugar uptake assays. 3. Synthesis of sugar and phlorizin based photoa ffinity probes for the identification of sugar translocation pathways and inhibitor binding sites. 4. Photoaffinity labeling of loop 13 with [(2’-Iodo-4’-(3’’ -trifluoromethyldiazirinyl) phenoxy]- D-glucopyranoside (TIPDG) , [(4’-Benzoyl) phenoxy)]- D-glucopyranoside (BzG), and 3-Azidophlorizin (3-AP). 5. Photoaffinity labeling of recombinant full-length hSGLT1. Materials and methods 14 3. Materials and methods Protocols were adapted from standard methods (Sambrook and Russell, 2001) unless otherwise mentioned. All solutions and media were prepared with ultra-pure water (Milli-Q Plus ultra purification pak, Milli-RO Plus purification pak; Millipore, Molsheim, France). 3 .1. General equipments Autoclave Autoclave 23; MELAG Medizintechnick (Berlin, Germany) Bioclav; Schütt Labortechnik GmbH (Göttingen, Germany) Centrifuges Biofuge pico, Omnifuge 2.OR, Megafuge 1.ORS, Heraeus (Osterode, Germany) Centrikon H- 401, with Rotors A8.24 and A6.9; Kontron-Hermle (Gosheim, Germany) Chromatography System Automated Econo System; Biorad (Hercules, CA) Electroporation apparatus Gene pulser II; Biorad (Hercules, CA) Freezer, -70 °C ULT1706; Revco Scientific (Asheville, NC), Gel documentation camera Polaroid MP-4 land camera; Polaroid USA Gel Electrophoresis Biorad (Hercules, CA) Incubators WTB Binder (Tuttlingen, Germany), Forma Scientific (USA) JULABO Labortechnik GmbH Liquid nitrogen tank BT40; L’air liquide (Champigny, France) Magnetic stirrer MR3001; heidolph (Kelheim, Germany) MALDI-MS PE-Biosystems, Shelton, USA Microscope IX50; Olympus Optical GmbH (Hamburg, Germany) Oriel photochemical Oriel, Stratford, CT, USA reactor Rayonet photochemical Southern New England, Branford, CT, USA reactor pH meter Model 765 Calimatic; Knick (Berlin, Germany) Scales Model BP 2100S; Sartorius (Göttingen, Germany) Sonicator SONOPLUS Bandelin electronic GmbH (Berlin, Germany) Spectrophotometer UVICON 930; Kontron instruments (Echingen, Germany) Thermocycler Mastercycler gradient; Eppendorf (Hamburg, Germany) Thermomixer Thermomixer compact; Eppendorf (Hamburg, Germany) Ultracentrifuge Optima TM TLX, with rotor TLA 100. 4; BeckMan Instruments Fullerton, CA Vortexer REAX top; Heidolph (Kelheim, Germany) Water baths GFL GmbH (Burgwedel, Germany) Materials and methods 15 Densitometer Pharmacia LKB (Sweden) UV illuminator Model N90M; UniEquip (Martinsried, Germany) 3 . 2 . Materials 3.2.1. Chemicals Acryl/Bis 37.5:1; 40% (w/v) Solution AMRESCO Inc. USA α- D-Acetobromoglucose Mo Bio Laboratories, USA Agar Mo Bio Laboratories, USA Agarose Sigma, Munich, Germany Ammonium persulphate Sigma, Munich, Germany Ampicillin Sigma, Munich, Germany Bromophenol blue Sigma, Munich, Germany 4-Bromoanisol Sigma, Munich, Germany BSA (bovine serum albumin) Sigma, Munich, Germany G250 Brilliant Blue Serva, Heidelberg, Germany α-Cyano-4-hydroxycinnamic acid Sigma, Munich, Germany Dideoxynucleotides Stratagene Inc USA 2,5-dihydroxybenzoic acid Sigma, Munich, Germany 3,5-Dimethoxy-4-hydroxycinnamic acid Sigma, Munich, Germany DMSO Sigma, Munich, Germany DTT Sigma, Munich, Germany EDTA Sigma, Munich, Germany Ethidium Bromide Sigma, Munich, Germany Formamide Fluka AG, Neu-Ulm, Germany FosCholine-12 Anatrace, Maumee, OH, USA D-[6- 3 H] Glucose Perkin-Elmer, LAS, Germany Glycerol Merck, Darmstadt, Germany HEPES Sigma, Taufkirchen, Germany 4-Hydroxybenzophenone Sigma, Munich, Germany IPTG peqLab Biotechnologie, Erlangen, Germany Methyl- α- D-[ 14C] glucopyranoside Perkin-Elmer, LAS, Germany SDS Sigma, Munich, Germany Sodium Azide Serva, Heidelberg Phlorizin Sigma, Munich, Germany TEMED Sigma, Munich, Germany Triton X-100 Sigma, Munich, Germany Tryptone Sigma, Munich, Germany Materials and methods 16 Tween 20 Sigma, Munich, Germany Yeast extracts Sigma, Munich, Germany Zeocin Invitrogen, Carlsad, USA All other Chemicals were of analytical grade and obtained from commercial sources. 3 . 2 . 2 . Enzymes and reagents for molecular biology Restriction enzymes New England Biolabs GmbH, Frankfurt, Germany Taq DNA polymerase New England Biol abs GmbH, Frankfurt, Germany Pfu Ultra HF DNA polymerase Stratagene, Netherlands Thermoscript reverse transcriptase Invitrogen GmbH, Karsruhe, Germany Calf Intestine alkaline phosphatase Roche Diagnostic, Mannheim, Germany T4 DNA ligase New England Biolabs GmbH, Frankfurt, Germany RNaseA Sigma, Munich, Germany DNase I Roche Diagnostic, Mannheim, Germany DNase (Rnase free) Qiagen, Hiden, Germany Lysozyme Sigma, Munich, Germany dNTP Stratagene, Netherlands T4 Polynucleotide kinase New England Biolabs GmbH, Frankfurt, Germany 3 . 2 . 3 . Antibodies Anti-FLAG M2 monoclonal antibody peroxidase conjugate Sigma, Munich, Germany Mouse anti-His-tag (27E8) monoclonal antibody Cell Signaling Technology Baverly, USA Rabbit anti-human SGLT1 antibody Acrirs Antibodies, Hiddenhausen, Germany 3 . 2 . 4 . Commercial Kits and other materials QIAquick TM PCR-Purification kit Qiagen, Hiden, Germany QIAquick TM Gel-extraction kit Qiagen, Hiden, Germany QIA ®Spin Miniprep kit Qiagen, Hiden, Germany QuickChange ® XL Site directed mutagensis kit Stratagene, Netherlands Rapid DNA ligation kit Roche Diagnostic, Mannheim, Germany Expend High Fidelity PCR system Roche Diagnostic, Mannheim, Germany Easy SelectTM Pichia Expression Kit Invitrogen, Carlsbad, USA Lowry Protein assay kit Sigma, Munich, Germany HybondTM-C extra blotting membrane Amersham Biosciences, Germany Materials and methods 17 SuperSignal®West Pico chemiluminiscent Pierce, Rockland, USA Substrate Photographic film Polapan; Polaroid, Offenbach, Germany 3 . 2 . 5 . Primers for preparation of cDNA inserts Primer Sequence Type, restriction site SGLT_F TATTGAATTCGATGGACAGTAGCACCTG Sense, EcoR1 SGLT_R AATACTCGAGGGCAAAATATGCATGGC Antisense, Xho1 ∆N12-377SGLT_F CTGCGAGGCCTGATGCTATCAGTCATGCTG Sense ∆N12-377SGLT_R GGTGGTCTTGGGGCTCCAGGTGCTACTGTC Antisense Spacer_His_SGLT_F GGTGGTAGCCCGGGTCACCACCACCACCAC CACTGAGAT Sense Spacer_His_SGLT_R GCTTTTATACAGCACGGCAAAATATGCATGG CAAAAGAC Antisense T7 promoter_F TAATACGACTCACTATAGGG Sense Unc-F_R TTTATTACAGTTCAGCGACAAG Antisense SGLT_FLAG_F GACGACGATAAGATCCAAGAAGGCCCTAAG GAGAC Sense SGLT_FLAG_R PBAD_SGLT_R ATCCTTGTAATCCAGGTCAATACGCTCCTCT TTGC TAATTAAGCTTTTAGGCAAAATATGCA TGG Antisense Antisense, Hind III pPICZ_SGLT_F TATTGAATTCAAAATGGACAGTAGCACCTG Sense, EcoR1 pPICZ α_SGLT_R ATTATTGCGGCCGCGGCAAAATATGCATGCC Antisense, Not1 3 . 2 . 6 . Plasmids 3.2.6.1. Original plasmids Plasmid Description Reference pET22b Expression vector, T7- lac promoter, Signal sequence at N-terminal of gene, at C-terminal His tag, Amp R, origin of replication pBR322. Novagen, San Diego, CA, USA pBAD24 P BAD promoter, His tag at C-terminal of gene, Amp R, pUC origin of replication. Invitrogen, Carlsbad, CA, USA pPICZB AOX1 promoter, His-tag at the C-terminal of gene, Zeo R; CoLE1 origin of replication. Invitrogen, Carlsbad, CA, USA pPICZ αA AOX1 promoter, α-factor secretion signal at the N-terminal of gene, His-tag at the C-terminal of gene, Zeo R; CoLE1 origin of replication. Invitrogen, Carlsbad, CA, USA Materials and methods 18 3.2.6. 2 . Plasmid with insert Plasmid Description Reference DKFZp686N20230Q2 Plasmid containing full-length hSGLT1 gene RZPD, Berlin, Germany pET22b-hSGLT1 hSGLT1 gene cloned in pET22b This study pET22b-hSGLT1- Spacer hSGLT1 gene cloned in pET22b with DNA sequence encoding for a 10 amino acids long spacer at the C-terminal of gene This study pET22b- ∆N12-377 hSGLT1 hSGLT1 gene cloned into pET22b after deletion of DNA sequence encoding amino acids 12-377 This study pET22b- ∆N12-377 hSGLT1-Spacer Clone pET22b- ∆N12-377 hSGLT1 with DNA sequence encoding for a 10 amino acids long spacer at the C-terminal of gene This study pET22b-Unc-F Unc-F gene encodes β -subunit of E. coli ATPase under the control of bacteriophage T7 promoter This study pET22b-Unc-F-hSGLT1 Unc-F gene cloned in pET22b plasmid encoding full-length hSGLT1 This study pET22b-Unc-F- hSGLT1-Spacer Unc-F gene cloned in pET22b plasmid encoding full-length hSGLT1 with C-terminal spacer This study pET22b-Unc-F- ∆N12- 377 hSGLT1 Unc-F gene cloned in pET22b plasmid encoding hSGLT1 gene shorten at N-terminal from 12-377 amino acids This study pET22b-hSGLT1- FLAG-Spacer pET22b vector containing full-length hSGLT1 gene with C-terminal spacer and FLAG tag at position D574 . This study pBAD24-hSGLT1- FLAG-Spacer pBAD24 vector containing full-length hSGLT1 gene with C-terminal spacer and FLAG tag at position D574 . This study pBAD24- ∆N12-377 hSGLT1-FLAG-Spacer pBAD24 plasmid encoding hSGLT1 gene shorten at N-terminal from 12-377 amino acids and FLAG tag at position D574 This study pPICZB-hSGLT1 P. pastoris plasmid pPICZB containing full- length hSGLT1 gene with FLAG tag at D 574 This study pPICZ αA-hSGLT1 P. pastoris plasmid pPICZ αA containing full- length hSGLT1 gene with FLAG tag at D 574 This study Materials and methods 19 3.3. Work with E .coli 3.3.1. E .coli strains Strain Genotype Source C41 (DE3) BL21 (DE3) derivative with uncharacterized mutations Avidis C43 (DE3) BL21 (DE3) derivative with uncharacterized mutations Avidis C43 (DE3) Rosetta C43 (DE3) and Rosetta derivative This study DH5α F−φ80dlacZ ∆M15 ∆(lacZYA-argF)U169 deoR recA1endA1 hsdR17(r k−, mk+ ) phoA supE44 λ−thi-1 Origami (DE3) ∆ (ara-leu)7697 ∆ lacX 74 ∆phoA PvuII phoR araD139ahpC galK rpsL F´[lac + lacIq pro] (DE3) gor522:: Tn 10trx (Kan R, StrR, TetR) Novagen RosettaTM (DE3) F− ompT hsdSB (r B- mB- ) gal dcm (DE3) pRARE (Cam R) Novagen XL10-Gold TetR ∆(mcrA)183 ∆(mcrCB-hsdSMR-mrr)173endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB lacIq Z ∆M15 Tn10 (Tet R) Amy Cam R] Stratagene TOP10F - F´proAB, lacI q , lacZ ∆M15, Tn10 (Tet R) mcrA, ∆(mrr- hsdRMS-mcrBC), φ80lacZ ∆M15, ∆lacX74, deoR, recA1, λ -araD139, ∆(ara-leu) 7697, galU, galK, rpsL, (str R), endA1, nupG Invitrogen 3 . 3 . 2 . Media for bacterial culture LB-medium 1.0% tryptone 0.5% yeast extract 1.0% NaCl pH 7.2 Terrific Broth 1.2% tryptone 2.4% yeast extract 0.4% glycerol 17 mM KH 2PO 4 72 mM K 2HPO 4 1.5% agar was added to the media for production of agar plates. The following concentrations of the antibiotics were used if required: Table 3.1 Antibiotic stock solutions and its working concentration. Antibiotic Stock solution Working concentration Ampicillin Chloramphenicol Zeocin Tetracycline 100 mg/ml in water 30 mg/ml in ethanol 100 mg/ml in water 5 mg/ml in ethanol 100 µg/ml 30 µg/ml 100 µg/ml 10 µg/ml Materials and methods 20 3.3.3. General E. coli were cultured at 37°C with LB medium or te rrific broth. Bacteria transformed with an antibiotic resistance-conferring plasmid were selectively propagated by supplementing the LB medium with respective antibiotic (see Table 3. 1). All solutions and supplements used for work with E. coli were autoclaved for 20 min at 121°C or filter-sterilized. Bacterial strains for long-term were stored at –70°C in LB medi um supplemented with glycerol to a final concentration of 15 %. 3 . 3 . 4 . Transformation of plasmid in E .coli 3.3.4.1. Preparation of competent cells by CaCl 2 method LB medium (100 ml ) was inoculated with an overnight culture of E. coli DH5α and grown at 37°C with shaking to an OD 600 of 0.4. This culture was transfer to sterile, disposable, ice-cold 50-ml plastic tubes and store on ice for 10 minutes. After incubation, cells were centrifuged at 4,000 rpm for 15 min at 4°C. The pellet was resuspended in 50 ml ice-cold 100 mM MgCl 2, incubated for 30 min on ice, and centrifuged at 4° C. The cells were resuspended in 2 ml ice- cold 100 mM CaCl2, incubated for 24 h at 4°C, supplemen ted with ice-cold 0.6 ml glycerol and 2.4 ml 100 mM CaCl2 and stored in 200µl aliquots at –70°C. 3 . 3 . 4 . 2 . Heat shock transformation of the plasmid Treatment of competent bacterial cells with a brief heat shock enables transformation of DNA. Plasmid DNA or a plasmid ligation reacti on (not more than 15ng or in a volume 2.5 µL ) was mixed with 50µl thawed competent bacteria and incubated for 30 min on ice. The bacteria were treated for 90s by heat shock of 42°C, pl aced on ice for 1-2 min, diluted with 950 µl LB medium and incubated for 1 h at 37°C with shak ing. An aliquot of 50-200 µl or resuspended pellet was spread on LB/antibiotic agar plate a nd incubated overnight at 37°C to select for transformed bacteria. In case of ligation mixture, the transformed cells were pelleted by centrifugation at 13,000 rpm for 30 seconds, and the pellet was resuspended in 100µl of LB medium. The whole suspension was spread on the agar plate. 3 . 4 . Work with P. pastoris 3.4.1. P. pastoris strains Strain Genotype Phenotype X-33 Wild-type Mut + GS115 his4 His- , Mut+ KM71H arg4 aox1::ARG4 Mut S, Arg + GS115/Albumin His4 Mut S GS115/pPICZ/lacZ his4 His - , Mut+ Materials and methods 21 3.4.2. Media for P. pastoris culture Low Salt LB (Luria-Bertani) Medium 1.0 % tryptone 0.5% yeast extract 1.0% NaCl pH 7.0 Low salt LB medium is needed for use with Zeocin antibiotic. Low salt LB agar plates were made by the addition of 1.5% agar before autoclaving. Yeast Extract Peptone Dextrose Medium (YPD or YEPD) % yeast extract 2% peptone 2% dextrose (glucose) Yeast Extract Peptone Dextrose Medium (YPD ± Zeocin) 1% yeast extract 2% peptone 2% dextrose (glucose) ± 2% agar ± 100 µg/ml Zeocin Buffered Glycerol/ Methanol complex Medium (BMGY and BMMY) 1% yeast extract 2% peptone 100mM KH2PO 4, pH 6.0 1.34% YNB 4× 10-5 % biotin 1% glycerol or 1% methanol Breaking Buffer 50 mM KH2PO 4, pH 6.0 Protease inhibitors cocktails 10% glycerol 3 . 4 . 3 . General P. pastoris was grown at 28-30 °C in YPD medium. Cells transformed with pPICZB-hSGLT1 plasmid were selectively propagated by supplementing the YPD medium with Zeocin antibiotic (see Table 3.1). For protein expre ssion pPICZB-hSGLT1 plasmid transformed cells were grown in BMGY medium supplemented with Zeocin. P.pastoris cells for long-term were stored at –70°C in YPD medium supplemente d with glycerol to a final concentration of 15 %. Materials and methods 22 3.4.4 . Electroporation of P. pastoris with plasmid pPICZB or pPICZB- hSGLT1 The purified DNA samples of pPICZB a nd pPICZB-hSGLT1 were linearized by PmeI digestion at 37 °C for 3 h and the linearized plasmids DNA were purified by enzyme removal column. YPD medium (100 ml) was inoculated in a 1-liter flask with 0.2 ml of overnight culture of P. pastoris strain GS115 and the cells were grown at 28 °C overnight to an OD 600 of 2-4. Overnight grown culture was centrifuged at 1500 × g at 4°C for 5 min. The supernatant was removed and the pellet was resuspended with 25 ml of ice-cold sterile water. The resuspended cells were centrifuged again as above and the pellet was resuspended in 8 ml of ice-cold sterile 1 M sorbitol. The cells were centrifuged as above and then the pellet was resuspended with 1 ml of ice-cold sterile 1 M sorbitol. Th e suspension of competent cells was kept on ice and used the same day. The suspension of competent cells (40 µl) and of linearized DNA (10 µl, 10 µg) were mixed and transferred to an ice-cold electroporation cuvette. The cuvette with the mixture of cells and DNA was incubated on ice for 5 min. The cells were pulsed at 1.5 kV, 50 µF and 200 Ω and 1 ml of ice-cold 1 M sorbitol was added to the cuvette. The cuvette contents were transferred to a sterile 15 ml tube and incubated at 30 °C without shaking for 2 h. 200 µl of these cells were plated on YPD medium with 100, 500, and 1000 µg/ml Zeocin and incubated at 30 °C for 2 days. 3 . 5 . Recombinant DNA processing and manipulation 3.5.1. Buffers and solutions TBE buffer (5X) Tris-base 54 g (Working solution 0.5X) boric acid 27.5 g EDTA 20 ml of 0.5 M, pH 8.0 Final volume 1 Liter TE 10mM Tris (pH 7.6), ImM EDTA (pH8.0) DNA loading buffer (6X) 0,25% (w/v) bromophenol blue 0,25% (w/v) xylene cyanol 30% (v/v) glycerol 50 mM EDTA Ethidium bromide 1% (w/v) in water DNA standard SmartLadder TM Eurogentec GmbH, Germany Materials and methods 23 3.5.2 . Restriction endonuclease digestion of DNA For preparative purposes, 1-5 µg DNA was digested with 1-20 U of restriction enzyme in a volume of 10-50 µl of reaction buffer. Comple te digestion was confirmed by agarose gel electrophoresis. For analytical purposes, 0.2-1 µg DNA were digested with 1-5 U enzyme in a volume of 10-20 µl of reaction buffer. In both cases digests were incubated for 3h at 37°C. Reaction buffers were supplied by the manufacturer. Enzymes were heat-inactivated as recommended by the supplier or removed by purification of the digested DNA by column chromatography. 3 . 5 . 3 . Purification of the digested DNA To inactivate and remove the proteins e.g. restriction enzymes, digested DNA was purified by using the QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions with slight modification. Buffers were provided in the kit and all centrifugation steps were carried out at 13,000 rpm at RT in a tabletop microcentrifuge. The 5 volume of the buffer PB and one volume of isopropanol were added to the DNA solution. The sample was then applied to QIAquick spin column (Qiage n) and centrifuged for 1 min in order to bind DNA to the column. For washing of DNA on the co lumn, 0.75 ml of buffer PE was added to the column and spun down for 1 min. The flow-through was removed and the column was centrifuged for an additional 1 min to remove the residual ethanol. The column was then placed into a clean 1.5 ml tube and the DNA wa s eluted by the addition of 30-50 µl of buffer EB (10 mM Tris/HCl, pH 8.5) or dH 2O to the column followed by centrifuging for 1 min. If needed the DNA could be concentrated by precipitating with ethanol. 3 . 5 . 4 . Ethanol precipitation of DNA 5 M NaCl solution was added to the DNA sample to a final concentration of 250 mM. Three volumes of ethanol (-20°C) were added to the solution and stored on ice for 30 min and then centrifuged at 13,000 rpm for 15 min at 4°C. Th e pellet was washed with one volume of ice- cold 70 % ethanol, recentrifuged for 5 min and dried at room temperature on desk. The DNA was dissolved in water or TE (pH 8) for 20 min at 37°C at an appropriate concentration. 3 . 5 . 5 . Dephosphorylation of linearized plasmid DNA by CIP In order to prevent self-ligation of vector ends in cloning strategies linearized plasmid DNA was treated with calf intestine alkaline phosphatase (CIP). CIP catalyzes the hydrolysis of 5'- phosphate residues to 5'-hydroxyl ends. Since T4 DNA ligase requires 5'-phosphate residues to catalyze new phosphodiester bonds, ligation is only possible between vector ends and inserts, but not between vector ends themselves. Dephosphorylation was carried out directly Materials and methods 24 following plasmid linearization. CIP was added to the digestion mixture at a concentration of 1 U per pmol linearized vector DNA. After 45 mi n incubation at 37°C, CIP and the restriction enzymes were removed as described previously. 3 . 5 . 6 . Ligation of DNA fragments Ligation was carried out using Rapid DNA lig ation kit according to the manufacturer’s instructions with slight modification. All the buffers were provided with the kit. The insert DNA was employed at a 2-5 molar excess rela tive to the linearized and dephosphorylated vector DNA. The vector DNA and insert mixture were diluted in dilution buffer to a final volume of 10µl. 10µl of ligation buffer was added in diluted DNA mixture. 5unit of T4 DNA ligase was added and mix thoroughly. The ligation reaction mixture was incubated 5-10 minutes at 15-25 °C. The 5µl of ligation reaction mixture was used directly for transformation of the 200µl competent cells. 3 . 5 . 7 . Miniprep: small-scale preparation of plasmid DNA Plasmid DNA was purified from bacteria culture s by alkaline lysis of the bacterial cells by using QIAprep Spin miniprep kit according to th e manufacture instructions. In brief, A small culture (10ml) of bacteria was grown in order to amplify in vivo the plasmid of interest. The bacteria were harvested by centrifugation at 13, 000 rpm for 1 min at room temperature. This pellet was resuspended in 250µl buffer P1 (50 mM Tis·Cl pH 8.0, 10 mM EDTA, 100 µg/ml RNase A). 250µl lysis buffer P2 (0.2 N NaOH, 1 % SDS) was added in resuspended pellet followed by gently mixing and incubated for 5 minutes at room temperature. It causes denaturation of the DNA by NaOH and of the bact erial proteins by SDS. The alkaline mixture was neutralized by 350µl of buffer N3 (contai ning guanidine hydrochloride, 3M potassium acetate, pH 5.5) causing reannealing of plas mid DNA and precipitation of SDS. The white precipitate, containing Proteins, chromosomal DNA, SDS and cell debris was removed by centrifugation for 10 minutes at 13,000 rpm while the plasmid DNA wasleft in the solution. The supernatant was passed through the spin column by centrifugation at 13,000 rpm for 1 minute. After washing of the column with 0.75ml buffer PE, the column was additionally centrifuged for 1 minute to remove residual buffer. The DNA was eluted by adding 50µl water or TE and centrifugation of the column at 13,000 rpm for 1 min. 3 . 5 . 8 . Large scale preparation of plasmid DNA 100 ml of bacterial culture was centrifuged at 4,000 rpm for 15 min at 4°C. The pellet was resuspended in 10 ml of P1 (50m M Tris.Cl, pH 8.0; 10mM EDTA; 100 µg/ml Rnase A). Resuspended pellet was lysed with 10 ml P2 (200mM NaOH, 1% SDS) by incubating for 5 Materials and methods 25 min at room temperature. The lysis was stopped by adding 10 ml ice-cold P3 (3M potassium acetate, pH 5.5) and the white precipitated mi xture was poured into the barrel of QIAfilter cartidge and incubated for 10 min. After 10 minutes, the plunger was inserted into the QIAfilter cartidge and filtered the cell lysate into the 50ml tube. The lysate was loaded on an equilibrated anion exchange column that was s ubsequently washed twice with 30 ml of buffer QC (1.0M NaCl, 50mM MOPS, pH7.0; 15% isopropanol). The bound DNA was eluted with elution buffer QN (1.6M NaCl, 50mM MOPS, pH 7.0; 15% isopropanol) by gravity flow. The eluted DNA was precipitated by adding 0.7 volum es isopropanol at room temperature and subsequent centrifugation at ≥15,000xg for 30 min at 4°C. The pellet was washed with 5 ml 70 % ethanol, re-centrifuged for 10 min, dried under vacuum and re-dissolved in 1ml of endothelial electroporation buffer or TE. 3 . 5 . 9 . Qunatification of DNA and RNA solutions The concentration of nucleic acid solutions was determined by spectrophotometry. The ultraviolet (UV) absorption was meas ured at a wavelength of 260 nm (OD 260 ) using a quartz cuvette of 1 cm width. For double-stranded DNA an OD 260 =1.0 corresponds to approximately 50 µg DNA/ml. For RNA an OD 260 = 1.0 corresponds to approximately 40 µg RNA/ml. In addition the OD 260 was measured to estimate the purity of the nucleic acid sample. A ratio OD 260 /OD 280 of significantly less than 1.8-2.0 indicates protein contamination. 3 . 5 .10. Agarose gel electrophoresis Agarose gel electrophoresis was used for analytical and preparative purposes. The method is based on the migration of the negatively charged DNA towards the anode in an electric field. The fragments migrate through the gel matrix at rates inversely proportional to the logarithm (lo g10) of the number of base pairs. DNA bands we re visualized within an agarose gel by staining with the intercalating fluorescent dye ethidium bromide and subsequent illumination under UV light. The length of a DNA fragment is determined by comparison of its mobility to that of DNA standards. For casting gel, 1- 2 % (w/v) agarose was melted in 0.5xTBE electrophoresis buffer and supplemented with 0.5 µg/ml ethidium bromide and cast in casting tray of desired size. The gel was placed in an electrophoresis tank and submerged in 0.5xTBE buffer. The DNA samples were mixed with DNA lo ading buffer and loaded into the gel wells. In addition 5 µl of a DNA standard was loaded in parallel with the samples. Horizontal electrophoresis was carried out at approximately 100 V. The stained gel was photographed under UV light. Materials and methods 26 3.5.11. DNA recovery from agarose gel For preparative purposes DNA fragments of interest were cut out from stained agarose gels with a razor blade under UV illumination. The gel slices were solubilized and DNA was purified by using the QIAquick Gel extraction kit (Qiagen) according to the manufacturer’s instructions. Agarose gel slices were weighed (= 1 volume) and dissolved each slice in 3 volumes of solubilization buffer QG by incubation for 10 mi n at 50°C. 1 volume isopropanol was added and the solution was applied to a silica-ge l QIAquick spin column. The column was centrifuged at 13,000 rpm for 1 min, washed w ith washing buffer PE and centrifuged again. The extra centrifugation was done to remove residual buffer. DNA was eluted by adding 30- 50µl water or TE on the column and centrifugation of the column at 13,000 rpm for 1 min. All the buffers were provided by the manufacturer. 3 . 5 .12. DNA sequencing The sequence of specific target regions in recombinant plasmid DNA was determined by a commercial sequencing service (Agowa GmbH Be rlin, Germany). The sequence data were verified on the basis of the corresponding fluorescence electropherogram and subjected to computer analysis. 3 . 5 .13. DNA amplification of hSGLT1 gene by PCR for cloning into different expression systems Primers for PCR amplification of SGLT1 cDNA were commercially synthesized (MWG Biotech, Ebersberg, Germany). They were designed corresponding to the DNA segment to be amplified, provided with restriction sites for endonuclease digestion. Pairs of primers were designed to have equivalent melting temperatures (Tm), calculated according to the formula 1 Tm [°C] = (A+T)·2 + (G+C)·4. The anneali ng temperature for each PCR was typically estimated by experimentally. For preparative DNA amplification as part of cloning strategies, High fidelity DNA polymerase was used in preference to Taq DNA polymerase, due to its 3' →5' proofreading exonuclease activity whic h minimizes the risk of nucleotide misincorporation during elongation. PCR reaction composition for DNA amplification was as follows: Template DNA 20-50ng Sense primer (forward) 100 pMoles Antisense primer (reverse) 100 pMoles 1 A, T, G, C: number of the 2’-deoxyribonucleosides adenosine (A), thymidine (T)guanosine (G) and cytidine (C) with in the primer sequence Materials and methods 27 dNTPs 200µM each PCR reaction buffer 1x DMSO 5-10% (Optional, to increase yield, specificity, consistency) DNA polymerase 1-2.5 units Final reaction volume 100µl (preparative PCR), 20-50 µl (analytical PCR) Th PCR amplified products of hSGLT1 gene were analyzed by agrose gel electrophorsis, they were further proceeding for cloning into their respective vectors. Thermal cycle parameters for PCR was as follows: Lid temperature 105 °C Initial denaturation 94 °C, 5 minutes Denaturation 94 °C, 30 seconds Annealing 50-55 °C (primer pair and template specific), 1minute Elongation 72 °C, 1 miunte/kb Cycles 30 Final elongation 72 °C, 10 minutes cDNA sequence encoding full length human SG LT1 was amplified by using plasmid DKFZp686N20230Q2 as a template and subcloned into the EcoR1 and Xho1 sites of the bacterial expression vector pET22b. Primers used for the amplification were SGLT1_F and SGLT1_R (see section 3.2.5). For cloning into pBAD24 bacterial expre ssion vector, pET22b-hSGLT1-FLAG-Spacer plasmid (see section 3.5.14.2 for detail) was used as a PCR template. The primers pPICZ_SGLT_F and PBAD_SGLT_R containing EcoR1 and HindIII restriction sites respectively were used for PCR amplification. For cloning hSGLT1 gene into eukaryotic expression system Pichia pastoris, pET22b- hSGLT1-FLAG-Spacer plasmid (see section 3.5.14. 2 for detail) was used as a PCR template. hSGLT1-FLAG-Spacer gene product contains a FLAG epitop at postion of 574 amon acid and a 10 amino acids spacer at the end of hSGLT1 gene. The primers used for PCR amplification were pPICZ_SGLT_F and pPICZ α_SGLT_R primer containing EcoR1 and Not1I restriction sites respectively 3 . 5 .14. Addition or deletion mutagenesis in hSGLT1 gene This method is a PCR-based site directed mutage nesis system that eliminates the necessity to subclone the amplified mutated DNA fragment. This procedure starts with a supercoiled, dsDNA vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by PfuUltra® high-fidelity DNA polymerase. On incorporation of the oligonucleotide primers, a mutated plasmid is generated. After temperature cycling, the product is treated with Dpn I. Dpn I is used to digest the parental DNA template and select for the synthesized DNA containing mutations. Since DNA isolated from most E. coli strains is dam methylated, it is susceptible to Dpn I digestion, that Materials and methods 28 will cut only fully or hemimethylated 5′ -G m6 ATG-3 ′ DNA sequences 2. The nicked vector DNA incorporating the desired mutations is then transformed into the commercial XL10-Gold cells3 . This method is very rapid and generates mutants with the efficiency greater than 80%. The specific primer design consideration was applied which are as follows. Both the primers must anneal to different strands of the plasmid. Primers should be >22 bases in length The mismatched portions should be at or near the 5´ end of one or both of the primers with 15 or more bases of correct sequence on the 3´ end. For deletion, the primers should be designed in frame of the coding sequence of the gene. The forward primer and reverse primer should be designed at 3 ′ and 5′end of the deletion sequence respectively. For addition of base pairs in the cDNA of hSGLT1 the additional base pairs should incorporated at the 5’end of the primers. One or both of the primers must be 5´ phosphorylated. T4 polynucleotide kinase used for the 5´ phosphorylation of an oligonucleotide primers. The reaction composition of the phosphorylation reaction is as follows Primer 1µl (100pmole) Kinase buffer 0.5µl (1x) T4 polynucleotide kinase 1µl (10 units) Water 2.5µl The reaction mixture was incubated for 1 hour. The 1.25µl of the reaction mixture was directly used for PCR. The mutant strands synthesis reaction was setup for PCR according to the manufacturer’s instructions. The PCR reaction composition and PCR cycling parameter were as follows: Template DNA 30ng Sense primer4 (forward) 125ng Antisense primer (reverse) 125ng dNTPs 200µM each or 1µl(supplied with the kit) PCR reaction buffer 1x Quik solution reagent 3 µl (supplied with the kit) Water was added to a final volume of 50µl Then 1µl of PfuUltra HF DNA polymerase was adde d and PCR was done with the following thermal cycling parameters. Lid temperature 105 °C Initial denaturation 94 °C, 2 minutes Denaturation 94 °C, 50 seconds Annealing 60 °C, 1minute 2 While plasmid DNA isolated from almost all the commonly used E.coli strains such as DH5α (dam + ) is methylated and is suitable for mutagenesis, plasmid DNA isolated from the exceptional dam - E.coli strains, including JM110 and SCS11, is not suitable. 3 The DH5α chemical competent cells prepared in our lab are also worked well with this protocol. 4 The primers were design according to the mutagenic primer design rules, which were given in instruction manual. Materials and methods 29 Elongation 68 °C, 1 miunte/kb Cycles 18 Final elongation 68 °C, 10 minutes After the PCR the reaction mixture was cooled to ≤ 3 7 °C on ice. 1µl of Dpn I was added directly to reaction mixture and kept for 2 hours for digestion at 37 °C. The PCR product was purified by PCR purification kit as described before. The 5µl of purified PCR product was ligated with rapid DNA ligation kit and transformed into DH5 α cells. The positive clone was confirmed by DNA sequencing. 3 . 5 .14.1. Addition of 30 base pairs long spacer at the C-terminal of hSGLT1 gene cDNA sequence encoding a 10 amino acids l ong spacer (VLYKSGGSPG) was introduced at the C-terminal of hSGLT1 gene. For the a ddition of spacer sequence, two primers (each containing 5 additional amino acids coding base pairs). The primers used for this mutation were Spacer_SGLT_His_F and Spacer_SGLT_His_ R (see section 3.2.5) and designed as shown in Figure 3.1. The resultant plasmid was designated as pET22b-hSGLT1-Spacer. Figure 3.1 Positions of the mutation primers in pET22b-hSGLT1 plasmid. 15 base pairs were added to each primers. After Ligation 30 bps were incorporated between hSGLT1 gene and His tag. 3 . 5 .14.2. Addition of FLAG tag in hSGLT-spacer gene For better immunological detection and to provide ease in the purification process, the FLAG epitope was introduced in hSGLT1 by mutating the native hSGLT1 sequence D 574 AEEEN to D574 YKDDDDK. In pET22b-hSGLT1-Spacer plasmid 21 base pairs endcoding the FLAG tag were inserted at the postion 574. During this mutation 15 base pairs encoding wildtype 5 amino acids (574-579) were deleted. The forward primer (SGLT1_FLAG_F) and reverse primer (SGLT1_FLAG_R) were designed simila rly as shown in Figure 3.1. The resultant plasmid was designated as pET22b-hSGLT1-FLAG-Spacer. 3 . 5 .14.3. Deletion of N-terminal half of hSGLT1 gene To construct N-terminal truncated hSGLT1 gene, the base pairs 34 to 1131 (Amino acids 12- 377) were deleted by deletion mutagenesis as described above. The primers ∆N12- 377_SGLT_F and ∆N12-377_SGLT_FR were designed with a similar approach as describe in section 3.5.14.1 following the primer design rules as described in section 3.5.14. Spacer_SGLT_His_R Spacer_SGLT_His_F pET22b pET22b 3’ 3’ 5’ hSGLT1 His tag 5’ Materials and methods 30 3.5.15. Addition of Unc-F gene at the N-terminal of hSGLT1 gene pAVD10 vector (Avidis, Saint-Beauzire, Fran ce) encodes for the intra membrane inducer Unc-F gene product (i.e. the β -subunit of the E. coli ATPase) under the control of the bacteriophage T7 promoter. Unc-F gene was subcloned into pET22b vector pAVD10 plasmid was digested with XbaI enzyme and purified on agarose gel. The purified Unc-F gene fragment was ligated into similarly treated dephosphorylated pET22b and pET22b-hSGLT1 plasmids. After ligation 5 µl of ligated mixture was transformed into DH5α cells and positive clones were identified by XbaI digestion. In order to further confirm the correct orientation of Unc-F gene in pET22b-Unc-F and pET22b-Un c-F-hSGLT1 clones PCR reaction of these clones were performed with following primer: T7 promoter, sense, 5´- TAATACGACTCACTATAGGG-3a´nd Unc-F pr imer, antisense, 5´-TTTATTACAGTTCAG CGACAAG-3.´ 3 . 6 . Protein expression and analysis 3.6.1. Growth conditions of E .coli for hSGLT1 expression Each clone was tested for protein expression in different conditions: Types of E. coli cells Clone name DH5 α Rosetta C41 C43 C43 Rosetta Expression conditions* pET22b- hSGLT1 Yes Yes Yes Yes Yes Condition 1, 2, 3, 4, 5 and 6 in Rosetta, C43 and C43R cells pET22b- hSGLT1-Spacer Yes Yes Yes Yes Yes Same pET22b- ∆N12- 377 hSGLT1 Yes Yes Yes Yes Yes Same pET22b- ∆N12- 377 hSGLT1 Spacer Yes Yes Yes Yes Yes Same pET22b-Unc-F Yes Yes No Yes Yes Same pET22b-Unc-F- hSGLT1 Yes Yes No Yes Yes Same pET22b-Unc-F- hSGLT1-Spacer Yes Yes No Yes Yes Same pET22b-Unc-F- ∆N12-377 hSGLT1 Yes Yes No Yes Yes Same pET22b- hSGLT1- FLAG-Spacer Yes Yes Yes Yes Yes Same Materials and methods 31 pBAD24- hSGLT1- FLAG-Spacer Yes Yes Yes Yes Yes Conditions 7 and 8 in Rosetta, C43 and C43R cells pBAD24- ∆N12- 377 hSGLT1- FLAG-Spacer Yes Yes Yes Yes Yes Same *Specification of different expression conditions; 1. LB medium, 1 mM IPTG, 37 °C growing temperature. 2. LB medium, 1 mM IPTG, 37 °C growing temperature with 45 min heat shock before induction of IPTG. 3. LB medium, 1 mM IPTG, 22 °C growing temperature with 45 min heat shock before induction of IPTG. 4. LB medium, 0.5 mM IPTG, 16 °C growing temperature with 45 min heat shock before induction of IPTG. 5. TB medium, 0.5 mM IPTG, 16 °C growing temperature with 45 min heat shock before induction of IPTG. 6. TB medium with 1 M sorbitol, 0.5mM IPTG, 22 °C growing temperature with 45 min heat shock before induction of IPTG. 7. LB medium, 0.2% L- (+) arabinose, 22 °C growing temperature. 8. LB medium, 0.2% L- (+) arabinose, 22 °C growing temperature with 45 min heat shock before Arabinose induction. 3 . 6 . 2 . Growth condition of P. pastoris for hSGLT1 expression In pilot experiments, single colonies of transformed cells were used to inoculate 2.5 ml of BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate, pH 7.0, 1.34% (w/v) yeast nitrogen base, 4 ×10-5 % (w/v) biotin, and 1% (v/v) glycerol). After 16-20 h of incubation at 30 °C, the cells were pelleted at 1000 × g and were resuspended in BMMY medium (BMGY medium in which the gl ycerol was replaced by 1% (v/v) methanol) to induce protein expression. The positive colonies for recombinant protein expression were identified by Western blot analysis by using anti-FLAG antibody (Sigma). The clone with the highest protein yield was selected for medium-scale protein expression in 2-liters flasks. In all subsequent preparations, the cells were harvested at ~24 h of methanol induction. 3 . 6 . 3 . hSGLT1 purification from P. pastoris Membranes were prepared from P. pastoris cells as described below in the functional analysis of recombinant hSGLT1 section. Peripheral prot eins and proteins adhering to the membrane were removed by washing with 4 M urea in breaking buffer containing protease inhibitor cocktail (Roche) at 4 °C for 2 h and resulting membranes were centrifuged at 100,000 × g at 4°C for 40 min. The stripped membranes were solublized in buffer TG [20 mM Tris.Cl, pH 8/1 M NaCl/20% (v/v) glycerol] supplemente d by 1.2% FosCholine-12 (Anatrace, Maumee, OH) and protease inhibitor cocktail, resulted membrane solution was shaken at 4 °C, overnight. After removal of insoluble fraction by ultracentrifugation at 100,000 × g for 40 min at 4°C, the clear supernatant was bound to the preequilibrated Protino Ni 2000 prepacked (Macherey-Nagel) polyhistidine-tag purification column at 4 °C, unbound proteins were removed by washing with 3 column volume of buffer TG plus 0.2% FosCholine-12. Recombinant hSGLT1 was eluted with 9 ml of buffer TG plus 0.2% FosCholine-12 Materials and methods 32 supplemented with 250 mM imidazole and protease inhibitor cocktail. The purified protein containing fractions were concentrated to 0.35 ml by ultrafiltration in Centricon YM100 devices (Millipore) and stored at -20 °C for further use. 3 . 7 . Protein analysis 3.7.1. Buffers and solutions SDS sample buffer (2x) 100 mM Tris·HCl (pH 6.8) 20 % (v/v) glycerol 4 % (w/v) SDS 0.01 % (w/v) bromphenol blue 10% β -mercaptoethanol acrylamide/bis-acrylamide 40% (w/v) acrylamide/bis-acrylamide ratio 37.5:1 (Amersco) Stacking gel buffer 0.5 M Tris-HCl, pH 6.8 Resolving gel buffer 1.5 M Tris-HCl, pH 8.8 APS 10% (w/v) APS in water, aliquots were stored at -20 °C SDS 10% (w/v) SDS in water Electrophoresis Buffer (10x) 30g Tris-base 142g glycine 10g SDS Water was up to 1 liter Transfer buffer 5.82g (48mM) Tris-base 2.93g (39mM) glycine 200ml methanol Water was added up to 1 liter TBST buffer 10mM Tris-base (pH 7.6) 150mM NaCl 0.05% (v/v) Tween 20 Blocking solution 5% blocking milk (BioRad) in TBST Phosphatase cocktail (1:100) 0.1% SDS Coomassie staining solution 0.25% coomassie Brilliant Blue R-250 10%Acetic acid 40% methanol in water (filtered) Coomassie destaining solution 10%Acetic acid 40% methanol in water Materials and methods 33 3.7.2 . Measurement of protein concentration 3.7.2.1. Enhanced alkaline copper (Lowry) protein assay The method is based on Peterson’s modificati on of the micro-Lowery method (Peterson, 1977). An alkaline cupric tartrate reagent (Low ery reagent) complexes with the peptide bonds of proteins and becomes reduced to cuprous (Cu + ). The Cu + as well as the R groups of tyrosine, tryptophan, and cysteine residues then react with the Folin reagent. The reagent reacts by first producing an unstable product which is slowly reduce to become molybdenum/tungsten blue color complex. The absorbance is read at a suitable wavelength between 500 nm and 800 nm (preferably 750nm) fo r spectroscopic quantification of proteins in aqueous solution. Lowry protein assay kit (Sigma) was used for protein quantification according to the manufacturer instructions. Using BSA stock solution (400 µg/ml), a set of protein standards with 0-300 µg/ml was prepared. The protein samples we re dilute to 1.0 ml with water. To each set of protein standards and samples, 1 ml of Lowry reagent was added and incubated for 20 minutes at room temperature. With rapid and immediate mixing, 0.5 ml of Folin & Ciocalteu’s phenol reagent was added in each tube and allowed color to develop for 30 minutes. The absorbance was measured at the wavelength of 750 nm in a spectrophotometer. Based on the absorption values of the protein standards a calibration curve was calculated and used for determination of the protein concentrations in the samples. All protein standards and samples were prepared in duplicate. 3 . 7 . 3 . SDS-PAGE Table 3.2 Compostion of staking and resolving gel. Components Stacking gel 5% (5ml final vol.) Resolving gel 10% (10ml final Vol) Resolving gel 12% (10ml final vol.) Water Acrylamide mix 1.5M Tris (pH8.8) 1.0M Tris (pH6.8) 10% SDS 10% ammonium persulphate TEMED 3.2ml 0.83 ml 0.63 ml 0.05 ml 0.05 ml 0.005 ml 4ml 3.3ml 2.5ml 0.1ml 0.1ml 0.004ml 3.3ml 4ml 2.5ml 0.1 ml 0.1 ml 0.004 ml Protein samples were resolved for analytical purposes by polyacrylamide gel electrophoresis (PAGE). The use of the anionic detergent s odium dodecyl sulfate (SDS) enables separation according to the molecular weight of the proteins. It binds to the polypeptides and confers a negative charge, which is in direct proportion to their size. The gel matrix is prepared by polymerization of acrylamide and N,N'-methylenebisacrylamide via free radicals. Initial radicals arise from chemical decay of ammonium persulfate (APS) catalyzed by N,N,N',N'- tetramethylethylenediamine (TEMED). The elect rophoresis towards the anode is carried out Materials and methods 34 in a discontinuous buffer system that first concentrates SDS-protein complexes within a stacking gel before they migrate into the resolving gel. The size of proteins is determined by comparing their mobility with that of a protein standard. The resolving gel solution was prepared, poured between two clean glass plates and overlaid with water or butanol. After polymerization, the top of the gel was washed with water and the residual water was removed by filter paper. Freshly prepared stacking gel solution was filled and allowed to polymerize. The plastic comb wa s inserted in poured stacking gel to prepare the wells for sample loading. After polymeri zation comb was removed and the wells were washed with water to remove un polymerized acrylamide Subsequently the gel was placed in a vertical electrophoresis apparatus filled with 1x electrophoresis buffer. SDS-PAGE protein samples were prepared by mixing protein samples with 2x SDS sample buffer in the ratio of 1:1, if not already prepared with 1x SDS sample buffer. The SDS-PAGE protein samples and a protein standard were denatured at 95 °C for 5 min and loaded onto the gel. Electrophoresis was carried out at 200 V. The resolving gel wa s subsequently subjected to western blot analysis or gel staining. 3 . 7 . 4 . Coomasie staining of the polyacrylamide gels Coomassie Brilliant Blue is an aminotriarylmethane dye that forms strong covalent cpmplexs with proteins, most probably by a combination of van der Waals forces and electrostatic interactions with NH3 + groups. The uptake of dye is approximately proportional to the amount of protein. The polyacrylamide gel was immersed in at least 5 volumes of the coomassie staining solution and placed on a slowly rotating plateform for 15- 30 minutes at room temperature. The gel was destained in coomassie destaining solution on a slowly rotating plateforms, changing the destaining solution three to four times. The rapid destaing can also achevied by keeping the staining gel in hot water (80°C). The ream ing background was removed by leaving the gel overnight in water. 3 . 7 . 5 . Western blot analysis Western blotting consists of the transfer of electrophoretically separated proteins from a SDS- PAGE gel to a nitrocellulose membrane by el ectoblotting. The membrane-immobilized target protein is identified by an appropriate primary antibody and a horseradish peroxidase (HRP)- conjugated secondary antibody directed agains t the primary antibody. Detection of the antigen-antibody-antibody complex occurs by HR P-mediated oxidation of the chemilumin escent substrate luminol, a cyclic diacylhydrazide. The reaction product exhibits an excited state which decays to ground state via a light emitting pathway, and is detectable by exposure of the membrane to an autoradiography film. Materials and methods 35 After SDS-PAGE, the gel, nitrocellulose me mbrane pads and electrode papers were equibilirated with pre chilled transfer buffer. The gel cassette was placed with the gray side down on a clean surface. The fiber pad, electroblotting papers (7papers on each side), gel and nitrocelloluse membrane were arranged in the cassette. The firmly closed cassette was put into the blotting module and then placed into the electroblotting buffer tank with ice cooling unit. The proteins were transferred to the membrane towards the anode at 200 mA for 1 h at 4°C in ice-cooled transfer buffer with continuous stirring. Non-specific binding site s on the blotted membrane were blocked by shaking in blocking solution for 2 h at room temperature. After blocking, the membrane was washed thrice with TBST for 5 minutes each wash. The washed membrane was sealed in polybag along with the primary antibody and incubated at 4°C for over night with an end-to- end shaking. The membrane was washed thrice for minutes each washing and incubated with the secondary antibody in blocking solution for 45 minutes. The membrane was washed thrice for 5 minutes each washing. The freshly prepared chemiluminescent substrate working solution was added to the surface of the membrane (approx. 0.2 ml/cm 2 ) and incubated for 4 min at room temperature. The membrane was wrapped in plastic foil and exposed in the darkroom for 10 s - 30 min to an autoradiographi c film that was subsequently developed in dark room conditions. 3 . 8 . Functional analysis of recombinant hSGLT1 Sugar uptake assays were carried out in membranes prepared from Pichia GS115 cells harboring pPICZB-hSGLT1 plasmid and with purified recombinant hSGLT1 reconstituted into liposomes. 3 . 8 .1. Membrane preparations for transport studies Cells cultured in BMMY medium were harvested at O.D 600 of 4-6/ml by centrifugation at 3000 × g at 4°C for 10 min, washed once with ice- cold breaking buffer (50 mM sodium phosphate, pH 7.4, 10% glycerol) and resusp ended in breaking buffer supplemented with protease inhibitor cocktail. An equal volume of acid- washed chilled glass beads (0.5 mm diameter) was added to the suspension, and cells were disrupted by vigorous vortexing ten times for 1 min, with intervening 1 min incubations on ice. Unbroken cells were removed by centrifugation at 2000 × g at 4°C for 5 min. Membranes were pelleted at 100,000 × g at 4°C for 30 min and resuspended in membrane suspension buffer (100 mM Mannitol, 20 mM HEPES- Tris, pH 7.4) with protease inhibitor cocktail. Aliquots were snap-frozen and stored at -80 °C. The protein concentration of the membrane preparation was determined by using a micro-BCA kit. Materials and methods 36 3.8.2 . Reconstitution of recombinant hSGLT1 Proteoliposomes were made using Triton X- 100 destabilized liposomes (Jung et al., 1998; Heiermann et al., 1999; Rigaud and Daniel, 2003) . Liposomes were composed of 90% (w/v) asolectin soy lecithin and 10% (w/v) cholestero l. 10 mg cholesterol and 90 mg asolectin soy lecithin were dissolved in 5 ml of chloroform in a beaker. The solvent was evaporated under a stream of argon to obtain a thin layer of dry lipids. Last traces of solvent were removed under vacuum in a desiccator overnight. Before each reconstitution the lipids were suspended in 5 ml of 100 mM potassium phosphate, pH 7.5/2 mM β -mercaptoethanol to yield a lipid concentration of 20 mg/ml and subsequently sonicated in argon atmosphere in a tip probe sonicator (until the suspension became slightly clear), and resulting liposomes were stored in liquid nitrogen. Triton X-100 was added at con centrations corresponding to the onset and or total solublization of lipids as determined by turbidity measurements (Rigaud et al., 1995). Detergent-destabilized liposomes were mixed with purified protein in a 400:1 (w/w) and incubated at room temperature under gentle agitation for 10 min. Detergent was removed by adding Bio-Beads SM-2 activated according to (Holloway, 1973) at a wet weight beads: detergent ratio of 6:1. After 1 h of incubati on at room temperature, fresh Bio-Beads were added, and incubation was continued for an additional hour. After the third addition of Bio- Beads, incubation was continued overnight at 4°C. Bio-Beads were removed by filtration on glass silk. Proteoliposomes were concentrated by centrifugation at 300,000 × g for 45 min and stored in liquid nitrogen. Proteoliposomes pelle t for sugar uptake assay were resuspended in 100 mM potassium phosphate, pH 7.5/2 mM β -mercaptoethanol for final protein concentration of 0.1 µg/ml. 3 . 8 . 3 . Transport assay of hSGLT1 in membranes and in proteoliposomes Sugar uptake by right-side-out membrane ve sicles was performed as described for E. coli right-side-out membrane vesicles (Jung et al., 1998; Turk et al., 2000). Proteoliposomes (preloaded with 100 mM potassium phosphate, pH 7.5/2 mM β - mercaptoethanol) were subjected to three sonication/freeze/thaw cycles before uptake assays at 22°C. Uptake was initiated by mixing 10 µl of proteoliposomes with 10 µl of transport buffer 2X (200 mM choline.Cl, 50 mM NaCl, 100 mM mannitol, 20 mM Tris, 20 mM HEPES, 300 mM KCl, 6 mM MgSO 4, 2 mM CaCl2) with methyl α- D- [ 14C] glucopyranoside ([ 14C] α-MDG). Each reaction was stopped by with 1 ml of ice-cold stop solution (10 mM Tris, 10 mM HEPES, 100 mM mannitol, 150 mM KCl, 50 mM choline.Cl, 50 mM NaCl, 3 mM MgSO 4, 1 mM CaCl2, 0.2 mM phlorizin), applied centrally to a 0.22 µm nitrocellulose filter GSWP (Millipore) over vacuum and washed with 3 ml of ice-cold stop solution, and the filter was assayed by scintillation counting. All experiments were performed at least in triplicate, and errors indicate the SE of the mean values. Materials and methods 37 3.9. Chemical synthesis of photoaffinity probes TIPDG, BzG, 3-AP and 6-AG we re synthesized according to schemes given in Figure 3.2, Figure 3.3, Figure 3.4 and Figure 3.5. 3 . 9 .1. [(2’-Iodo - 4 ’ - (3’’-trifluoromethyldiazirinyl) phenoxy]- D- glucopyranoside (TIPDG) 3.9.1.1. 4-Methoxytrifluoroacetophenone (1) The titled compound in (Figure 3.2) was synthesized as previously reported (Delfino et al., 1993; Hatanaka et al., 1994) in 95% yield. 3 . 9 .1.2. 3-Iodo-4-methoxytrifluoroacetophenone (2) A mixture of 10 g (0.5 mol) of 1, 21.6 g (1.0 mol) of HgO, 5 ml of concentrated H 2SO 4, and 12.7 g (0.5 mol) of iodine in 250 ml of CCl 4 was heated to reflux with vigorous stirring for 4 h. The reaction mixture was cooled to room temperature, and then filtered through a pad of celite. The filtrate was washed with 2 N aqueous sodium thiosulfate solution and brine, and dried over Na2SO 4. The residue obtained after evaporation was purified by column chromatography (hexane: EA = 9:1) to give 13.87 g (85%) of 2 as a white solid; mp 52-54ºC. 1H NMR (CDCl 3 , 400MHz): δ = 3.98 (s, 3H), 6.89 (d, 1H, J = 8.8Hz), 8.04 (d, 1H, J = 8.8Hz), 8.47 (s, 1H). 13 C NMR (CDCl 3, 100MHz) : δ = 56.9, 86.5, 110.3, 116.6, 124.3, 132.7, 141.7, 163.6, 177.9. 3 . 9 .1.3. 4-Hydroxy-3-iodo-trifluoroacetophenone (3) O -Demethylation of 2 was carried out as follows. A mixtur e of 6.70 g (20 mmol) of 2 and 2.6 g (62 mmol) of LiCl in 50 ml of DMF was heated at reflux under argon for 2 h. The reaction mixture was cooled to room temperature, poured into water (200 ml) and acidified with 10% HCl. The product was extracted with Et2O .The ethereal layer was washed with brine twice, dried over MgSO 4, and the residue obtained after evaporation of solvent was purified by column chromatography (CH 2Cl2) affording 5.59 g (88%) of 3 as a white solid; mp 87-88ºC. 1H NMR (CDCl 3 , 400MHz): δ = 6.13 (s, 1H), 7.09 (d, 1H, J = 8.8Hz), 7.98 (d, 1H, J = 8.6Hz), 8.40 (d, 1H, J = 0.4Hz). 13 C NMR (CDCl 3 , 100MHz): δ = 86.3, 115.3, 118.0, 124.6, 125.9, 132.9, 141.2, 160.8, 178.4. DEI-MS 316 (M + ); HRMS calcd for (C 8 H4F3 IO 2) + 315.9208, found 315.9208. 3 . 9 .1.4. [(2´-Iodo-4´-trifluoroacetyl) phenoxy]-2,3,4,6-tetra-O-acetyl- D- glucopyranoside (5) To a solution of α- D-acetobromoglucose 4, 4.0 g (9.73 mmol) in 80 ml of dry benzene-nitro methane 1:1, 3.07 g (9.71 mmol) of compound 3, 2.45 g (9.73 mmol) mercuric cyanide, and 3.0 g molecular sieves (4Å) were added. The resulting suspension was stirred for 12 h at 50ºC. Materials and methods 38 The solvent was evaporated in vacuum and the residue was purified by column chromatography (EA: P.E = 3:1) to yield 4.71 g (75%) compound 5. 1H NMR (CDCl 3 , 400MHz): δ = 2.01, 2.03, 2.07, 2.09 (s, 3H, H-Ac), 4.01-4.16 (m, 1H, H-5), 4.20-4.28 (m, 2H, H-6), 4.88 (dd, 1H, J1, 2 = 4.5Hz, H-1), 5.07 (t, 1H, J = 11.7Hz, H-4), 5.18-5.33 (m, 1H, H-2), 5.53 (t, 1H, J3, 4 = 11.7Hz, H-3), 7.03 (d, 1H, J = 10.75Hz, Ar-H), 7.92-7.96 (m, 1H, Ar-H), 8.41 (s, 1H, Ar-H). 13 C NMR(CDCl 3 , 100MHz): δ = 21.05, 21.16, 21.17, 21.23, 62.40, 67.43, 68.87, 70.27, 71.46, 95.73, 115.36, 118.25, 124.39, 132.84, 141.81, 161.71, 169.92, 170.41, 170.49, 171.19, 177.57, 177.70, 178.05, 178.83. FAB-MS 646.09 (M-H) - , 670.04 (M+Na) + , HRMS calcd for (C 22H23 O 11F3 NaI) + 670.0134, found 670.0122. 3 . 9 .1.5. [(2´-Iodo-4´-trifluoroacetyloxime) phenoxy]-2,3,4,6-tetra-O-acetyl- D- glucopyranoside (6) A solution of 5, 4.0 g (6.18 mmol) and hydr oxylamine hydrochloride 1.27 g (18.28 mmol) in 10 ml absolute ethanol and 30 ml dry pyridine wa s stirred at 60ºC for 8 h. After evaporation of the solvent, the residue was partitioned between water and ether. The organic layer was washed with 1 N HCl and dried over MgSO 4. After evaporation of the solvent, the crude oxime was purified by column chromatography on silica gel (CH 2Cl2: MeOH = 10:1) to leave 3.68 g (90%) of a colorless syrupy product 6. 1H NMR (CDCl 3 , 400MHz): δ = 2.01, 2.04, 2.06, 2.09 (s, 3H, H-Ac), 4.09-4.15 (m, 1H, H-5), 4.22-4.30 (m, 2H, 2H-6), 4.88 (dd, 1H, J1, 2 = 4.5Hz, H-1), 5.08 (t, 1H, J = 11.7Hz, H-4), 5.20-5.34 (m, 1H, H-2), 5.54 (t, 1H, J3, 4 = 11.7Hz, H-3), 7.04 (d, 1H, J = 10.75Hz, Ar-H), 7.92-7.96 (m, 1H, ArH), 8.36 (s, 1H, NOH), 8.41 (s, 1H, ArH). 13 C NMR (CDCl 3 , 100MHz): δ = 21.06, 21.15, 21.18, 21.24, 62.33, 67.36, 68.89, 70.33, 71.49, 95.73, 115.38, 118.25, 124.40, 132.84,141.41, 141.83, 161.71, 169.92, 170.49, 177.58, 177.70, 178.15, 178.83 . FAB-MS 661.96 (M+H) + , 683.94 (M+Na) + , HRMS calcd for (C 22H24NO 11F3 I) + 662.0347, found 662.0356, calcd for (C 22H23 NO 11F3 NaI) + 684.0165, found 684.0197. 3 . 9 .1.6. [(2´-Iodo-4´-(3´´-trifluoromethyldiazi ridinyl) phenoxy]-2,3,4,6-tetra-O-acetyl- D- glucopyranoside (7) To a solution of oxime 6, 3.5 g (5.28 mmol), triethylamine 1.44 g (10.54 mmol) and ( N,N- dimethylamino) pyridine 25 mg (0.02 mmol) in 20 ml CH 2Cl2 at 0ºC, was added p - toluenesulfonyl chloride 1.0 g (5.28 mmol) porti on wise with stirring. After the addition, the reaction mixture was stirred at room temperature for 45 min. The mixture was washed with water, and the organic phase was dried over MgSO 4. After evaporation of the solvent, the crude oxime tosylate was dissolved in 20 ml dry CH2Cl2, and the solution is cooled to –78 ºC in a sealed tube. Liquid ammonia 3 ml was added and the mixture was stirred at room temperature for 16 h in a sealed tube. The exce ss ammonia was allowed to evaporate at room temperature. The residue was partitioned between water and CH2Cl2, and the organic layer was dried over MgSO 4. After evaporation of the solven t, the residual syrupy product was purified by column chromatography on neutral alumina (CH 2Cl2) to give 2.27 g (65%) of a Materials and methods 39 pale yellow syrupy compound 7. 1H NMR (CDCl 3 , 400MHz): δ = 2.00, 2.01, 2.06, 2.08 (s, 3H, H-Ac), 4.00- 4.28 (m, 5H, H-5, H-6, NH), 4.78 (dd, 1H, J1, 2 = 4.4Hz, H-1), 5.02 (t, 1H, J = 11.6Hz, H-4), 5.12-5.27 (m , 1H, H-2), 5.43 (d, 1H, J 3, 4 = 11.7Hz, H-3), 7.02 (d, 1H, J = 10.75, Ar-H), 7.90-7.96 (m, 1H, Ar-H), 8.41 (s, 1H, Ar-H). FAB-MS 659.73 (M) + , 661.06 (M+H) + , 683.08 (M+Na) + , HRMS calcd for (C 22H24N2O 10F3 I) + 661.3408, found 661.0506, calcd for (C 22H24N2O 10F3 NaI) + 683.3226, found 683.0297 3 . 9 .1.7. [(2´-Iodo-4´-(3´´-trifluoromethyldia zirinyl) phenoxy]-2,3,4,6-tetra-O-acetyl- D- glucopyranoside (8) To a vigorously stirred solution of 7, 1.0 g (1.51 mmol) and triethylamine 0.3 g (3.0 mmol) in 10 ml MeOH was added portionwise I 2 total 0.39 g (1.54 mmol). After being stirred for 30 min at room temperature, the dark brown reaction mixture was partitioned between Et2O and saturated aqueous citric acid. The Et 2O phase was washed with soluti ons of citric acid, sodium hydrogen sulfite and finally with water. The organic layer was dried over MgSO 4 and concentrated in vacuo to give syrupy residue, which was purified by column chromatography on silica (CH 2Cl2) to give 0.9 g (91%) yellow color syrupy compound 8. 1H NMR (CDCl 3 , 400MHz): δ = 2.00, 2.01, 2.06, 2.08 (s, 3H, H-Ac), 4.00-4.10 (m, 1H, H-5), 4.18-4.24 (m, 2H, H-6), 4.76 (dd, 1H, J1, 2 = 4.4Hz, H-1), 5.02 (t, 1H, J = 11.6Hz, H-4), 5.12-5.26 (m, 1H, H-2), 5.40 (d, 1H, J 3, 4 = 11.7Hz, H-3), 7.00 (d, 1H, J = 10.75, Ar-H), 7.90-7.96 (m, 1H, Ar-H), 8.40 (s, 1H, Ar-H). FAB-MS 658.00 (M) + , 681.04 (M+Na) + , HRMS calcd for (C 22H22O 10N2F3 NaI) + 681.0168, found 681.0159. Figure 3.2 Reaction scheme for the synthesis of TIPDG 9. The reagents were (a) I 2, HgO, conc. H 2SO 4, CCl4; (b) LiCl, DMF; 3,4- hydroxy-3-iodo-trifluor oacetophenone; (c) Hg(CN) 2, nitro methane, benzene; (d) NH2OH.HCl, EtOH, pyridine; (e ) tosyl chloride, DMAP, Et 3 N, DCM, NH3 ; (f) I 2, Et3 N, MeOH; (g) NaOMe. Materials and methods 40 3.9.1.8. [(2´-Iodo-4´-(3´´-trifluoromethyldiazirinyl) phenoxy]- D-glucopyranoside (9) Compound 8, 0.5 g (0.75 mmol) was deacylated by stirring with 10 ml dry MeOH containing 0.9 g (22.5 mmol) NaOH for 2 h at room temper ature. The solution was neutralized with ion exchange resin (Amberlite IR 120, H + ), the solvent was evaporated in vacuo and the residue was purified on a silica column (CHCl 3 : MeOH = 9:1) to yield 0.36 g (98%) 9, λmax (ME) = 358 nm ( ε = 290 M -1 .cm-1 ). 1H NMR (CD 3 OD, 400MHz): δ = 3.25 (dd, 1H, J = 7.7Hz,H-2), 3.30-3.39 (m, 3H, H-3, H-4, H-5), 3.70-3.82 (dd, 1H, J = 6.0Hz, H-6), 3.88-3.91 (dd, 1H, J = 1.6Hz, H-6), 4.27 (d, 1H, J = 7.8Hz, H-1), 7.06 (d, 1H, J = 10.8, Ar-H), 7.94-7.98 (m, 1H, Ar- H), 8.41 (s, 1H, Ar-H). 13 C NMR (CD 3 OD, 100MHz): δ = 71.51, 71.56, 75.15, 78.03, 78.15, 103.0, 117.37, 120.52, 126.92, 134.82, 171.20, 176.67, 178.07, 179.05, 179.93.FAB-MS 489.98 (M) + , 490.99 (M+H) + , HRMS calcd for (C 14H14N2O 6 F3 I) + 489.9846, found 489.9846, calcd for (C 14H15N2O 6 F3 I) + 490.9925, found 490.9927. 3 . 9 . 2 . [(4´-Benzoyl) phenoxy ]-D- glucopyranoside (BzG) 3.9.2.1. [4´-Benzoyl) phenoxy] –2,3,4,6-tetra-O-acetyl- D- glucopyranoside (11) A solution of α- D-Acetobromoglucose 4, 5.0 g (12.16 mmol) in 50 ml of benzene and in 50 ml of nitro methane was mixed with 4-hydroxybenzophenone 10, 2.41 g (12.16 mmol), mercuric cyanide 3.07 g (12.16 mmol) and 3.9 g of molecular sieves (4Å) and the resulting mixture was stirred at 60ºC for 15 h. The reac tion mixture was partitioned in between water and Et2O and the resulting organic phase was wa shed with brine and dried over MgSO 4. After evaporation of the solvent in vacuo, the resulting residue was purified by column chromatography on silica (EA: PE = 1:1) to yield 4.36 g (68%) white solid compound 11. 1H NMR (CDCl 3 , 400MHz): δ = 2.01, 2.02, 2.06, 2.07 (s, 3H , H-Ac), 4.08-4.11 (m, 1H, H-5), 4.21-4.26 (m, 2H, H-6), 4.88 (dd, 1H, J1, 2 = 4.5Hz, H-1), 5.06 (t, 1H, J = 11.7Hz, H-4), 5.20- 5.28 (m, 1H, H-2), 5.53 (t, 1H, J3, 4 = 9.4Hz, H-3), 6.80 (dd, 2H, J = 8.42 and 2.45Hz, Ar-H), 7.43-7.47 (m, 2H, Ar-H), 7.52-7.56 (m, 1H, Ar-H), 7.70-7.75 (m, 4H, Ar-H). 13 C NMR (CDCl 3 , 100MHz): δ = 21.08, 21.16, 21.18, 21.22, 67.34, 68.89, 70.36, 71.51, 95.67, 115.51, 128.42, 129.55, 132.27, 133.23, 138.26, 161.15, 169.99, 170.50, 170.58, 171.30, 196.61. 3 . 9 . 2 . 2 . [(4´-Benzoyl) phenoxy]- D- glucopyranoside (12) Deacetylation of compound 11 was carried out as follows. A mixture of 2.0 g (3.78 mmol) of 11 and 4.53 g (151.2 mmol) NaOH in 20 ml dry methanol was stirred at room temperature for 5 h. After completion of reaction the resulting reaction mixture was neutralized with ion exchange resin (Amberlite IR 120, H + ), the solvent was evaporated in vacuo and crude product was purified by column chromatography on silica (CHCl 3 : MeOH = 3:1) to yield 1.22 g (90%) white solid compound 12; mp 163 ºC; λmax (ME) = 293 nm ( ε = 290 M -1 .cm-1 ). 1H Materials and methods 41 NMR (CD 3 OD, 400MHz): δ = 3.22 (dd, 1H, J = 7.6Hz, H-2), 3.27-3.38 (m, 3H, H-3, H-4, H- 5), 3.68-3.71 (dd, 1H, J = 5.9Hz, H-6), 3.87-3.90 (dd, 1H, J = 1.6Hz, H-6), 4.27 (d, 1H, J = 7.8Hz, H-1), 7.51 (t, 2H, J = 7.8Hz, Ar-H), 7.62 (tt, 1H, J = 1.7 and 6.4Hz, Ar-H), 7.69 (d, 2H, J = 8.1Hz, Ar-H), 7.83 (m, 4H, Ar-H). Figure 3.3 Synthesis of BzG 12. The reagents were (a) Hg(CN) 2, nitro methane, benzene; (b) NaOMe. 3.9.3. 3-Azidophlorizin (3-AP) 3.9.3.1. 3-Nitrophlorizin (14) 472 mg Phlorizin 13 was dissolved in 10 ml of glacial acetic acid. The temperature of the solution was brought to 15°C. 75 µl of nitric acid (specific gravity 1.4) was added dropwise to the solution under vigorous stirring. At the end of the reaction, the color of the solution turned to red-brown. The solution was then poured onto 20 g ice. The green precipitates were collected and dried under vacuum. For further purification, the dry precipitates were dissolved in tetrahydrofurane. The solution was passed through a neutral aluminum oxide column. The yellow solution was then concentrated and a small amount of petroleum ether 30-60 °C was added until the solution became slightly turbid. After some time crystals developed. Melting point 217-218 °C. 3 . 9 . 3 . 2 . 3-Aminophorizin (15) 482 mg 3-Nitrophlorizin 14 was dissolved in 5 ml ethanol and mixed with 150 mg palladium/carbon (10% Pd). Hydrogen gas was introduced into the solution gently for a period of 1 h at room temperature. The catalyst was then removed by filtration. The ethanolic solution was diluted with 10 ml of deoxygenated water and frozen immediately. Solid 3- Aminophlorizin was obtained by freeze-drying. The yield of reduction product was almost 100%. Melting point 91-93 °C. During the whole procedure the reaction mixture was protected from light by aluminum foil. Materials and methods 42 Figure 3.4 Reaction scheme for the synthesis of 3-AP. The reagents were (a) HNO 3 , CH3 COOH; (b) H 2, Pd/C, EtOH; (c) NaNO 2, NaN3 . 3 . 9 . 3 . 3 . 3-Azidophlorizin (16) 45 mg of 3-Aminophlorizin were dissolved in 3 ml of 0.5 N ice-cold hydrochloric acid. The solution was cooled down to -5 °C. To this solution 0.5 ml 0.2 M cold sodium nitrite was added. The reaction was allowed to proceed at -5 °C for 10 min. A deep green color developed. Subsequently, 0.5 ml of 0.2 M sodi um acetate was added. The solution changed its color rapidly to red brown while bubbling occurred because of nitrogen release. A gel-like precipitate was obtained by centrifugation (low sp eed). The dry precipitate was then dissolved in an excess of THF. The deep brown solution was decolored by passing through a short silica gel column. After part of THF had been eva porated, a few drops of water were added to obtain 3-Azidophlorizin precipitate. Melting point 85 °C (decomposed). 3 . 9 . 4 . n-Methyl-6C- (Azimethyl)- D- glucopyranoside (6-AG) 3.9.4.1. Methyl-2,3,4-tri-O-benzyl-7-deoxy- DL-glycero-D-glucohepta-1,5- pyranoside (18) Compound 17, 11.5 g (25 mmol) (Lehmann and Th ieme, 1986) dissolved in 200 ml dry ether was added at room temperature over a period of 45 min to a stirred solution of methylmagnesium iodide prepared from 5.4 g (0.18 mol) magnesium turnings and 24.8 g (0.18 mol) methyl iodide in 310 ml dry ethe r. The mixture was stirred for 1 h at room temperature and poured on 400 g ice, and saturated aqueous NH 4Cl was added until Materials and methods 43 precipitated magnesium salts dissolve. The organic phase was separated and the aqueous layer was extracted three times with 150 ml ether. The combined ether extracts were washed with water, dried with MgSO 4, evaporated in vacuo to yield 11.5 g (97%) as syrup. The crude product was used without further purification in next step. 3 . 9 . 4 . 2 . Methyl-2,3,4-tri-O-benzyl-7-deoxy- D-glucohepto-1,5-pyranoside-6-ulose (19) Compound 18, 11.5 g (24 mmol), 14.0 g (69 mmol) DCC, and 1.0 g anhydrous phosphoric acid were dissolved in 170 ml anhydrous dimethyl sulxfoside and the mixture was stirred for 19 h at room temperature. The suspension was added slowly with stirring to an ice-cold solution of 9.5 g (0.11 mol) oxalic acid in 20 ml methanol within 15 min; the mixture was stirred for 30 min and diluted with 350 ml saturated aqueous NaCl. After stirring for a further 30 min, the precipitated solid was filtered and s tirred two times with 250 ml ether and filtered again. The combined ether extracts were neutralized with saturated aqueous NaHCO 3 , washed with water, dried with MgSO 4, and evaporated in vacuo. The syrupy crude product was dissolved in boiling ethyl acetate (40 ml) and af ter addition of 40 ml petroleum ether it was crystallized at -20 °C to yield 5.5 g (48%) 19, melting point 97 °C. 1H NMR (CDCl 3 , 500MHz): δ = 2.16 (s, 3H, H-7), 3.39 (s, 3H, OCH 3 ), 3.54 (dd, 1H, H-2, J2, 3 = 9.6Hz), 3.62 (t, 1H, H-4, J4, 5 = 9.9Hz), 4.01 (t, 1H, H-3, J 3 , 4 = 9.5Hz), 4.12 (d, 1H, H-5), 4.61 (d, 1H, H-1, J1, 2 = 3.5Hz), 4.60-5.00 (m, 6H, H-Bn), 7.72-7.36 (m, 15H, H-Ph). 13 C NMR (CDCl 3 , 125.8MHz): δ = 27.85, 55.55, 74.22, 73.46, 74.84, 75.79, 78.59, 79.57, 81.77, 98.53, 127.58-128.43, 137.84, 137.96, 138.55, 204.36. 3 . 9 . 4 . 3 . Methyl- 7- deoxy- D-glucohepta- 1, 5- pyranoside-6- ulose (20) Compound 19, 5.8 g (12 mmol) dissolved in 200 ml methanol-ethyl acetate was debenzylated in the presence of 3.0 g palladium (10% C) by catalytic hydrogenation for 5 h with shaking using a hydrogen pressure of 3 bar at room temp erature. The suspension was filtered from the catalyst and evaporated in vacuo to yield 2.5 g (100%) 20 as syrup. For further characterization 0.86 g (4.2 mmol) 17 was per-O-acetylated by stirring with 30 ml pyridine-acetic anhydride (2:1) for 15 h at room temperature. The mixture was poured on 250 g ice, stirred for 30 min, and extracted three tim es with 50 ml dichloromethane. The combined organic extracts were washed with water, dried with MgSO 4, and concentrated in vacuo. The resulting syrup was purified on silica gel column to yield 0.97 g (67%) acetylated compound 21. 1H NMR (CDCl 3 , 500MHz): δ = 2.01, 2.02, 2.09 (s, 3H, H-Ac), 2.26 (s, 3H, H-7), 3.45 (s, 3H, OCH 3 ), 4.09 (d, 1H, H-5), 4.89 (dd, 1H, H-2, J2, 3 = 10.2Hz), 5.03 (d, 1H, H-1, J1, 2 = 3.6Hz), 5.10 (t, 1H, H-4, J4, 5 = 10.3Hz), 5.50 (t, 1H, H-3, J 3 , 4 = 9.8Hz). 13 C NMR (CDCl 3 , 125.8MHz): δ = 20.51- 20.63, 25.82, 55.85, 68.93, 69.47, 70.72, 73.32, 97.03, 169.64, 169.79, 170.09, 203.65. Materials and methods 44 3.9.4 . 4 . n-Methyl-6C-(Azimethyl)- D-glucopyranoside (22) A stream of dry NH 3 was introduced into a solution of 2.5 g (12 mmol) 20 in 100 ml dry methanol at -30 °C until the volume increased about 20% . Then 2.0 g (17 mmol) hydroxyl-O- sulfonic acid was added in small portions over a time period of 1 h to the vigorously stirred mixture. The suspension was stirred for 3 h at -30 °C, allowed to attain room temperature overnight, filtered, and concentrated in vacuum. A solution of the residue in 100 ml dry methanol containing 20 ml Et3 N was cooled to 0°C and iodine was added until the red color persisted. The solution was then concentrated and the residue was purified on a silica gel column to give 1.6 g (61%) pure compound 22. λmax (Methanol) = 335 nm ( ε = 52 M -1 . cm-1 ). For further characterization of compound 22, 0.2 g of it was per-O-acetylated as described for compound 20, to yield 0.16 g (76%) compound 23. 1H NMR (CDCl 3 , 500MHz): δ = 1.08 (s, 3H, H-7), 2.02, 2.07, 2.15 (s, 3H, H-Ac), 3.02 (d, 1H, H-5), 3.39 (s, 3H, OCH 3 ), 4.82 (dd, 1H, H-2, J2, 3 = 10.2Hz), 4.96 (d, 1H, H-1, J1, 2 = 3.6Hz), 5.03 (t, 1H, H-4, J4,5 = 10.3Hz), 5.37 (t, 1H, H-3, J 3 , 4 = 10.1Hz). 13 C NMR (CDCl 3 , 125.8MHz): δ = 15.15, 20.47-20.83, 25.09, 55.33, 68.58, 69.52, 70.48, 71.19, 96.56, 169.54, 169.72, 169.90. Figure 3.5 Synthesis of 6-AG. The reagents were (a) MeMgI, H 2O; (b) Me 2SO, N, N´- dicyclohexylcarbodiimide; (c) H 2, Pd/C, EtOH; (d) Ac 2O, pyridine; (e) NH 3 , hydroxylamine-O-sulfonic acid, I 2; (f) Ac 2O, pyridine. Materials and methods 45 3.10. Photoaffinity labeling of loop 13 Truncated loop 13 protein used in this study was a gift from Mobeen Raja. Before using these photoprobes as a label for truncated loop 13 we performed transport assays of these probes with rabbit small intestine brush border membrane vesicles. 3 .10.1. Isolation of small intestine brush border membrane vesicles (BBMV) Rabbit small intestines were obtained immediately after sacrifice of the animals. Brush border membrane vesicles (BBMV) from rabbit small in testine epithelial scratching were prepared by the magnesium precipitation method of Booth and Kenny (Booth and Kenny, 1974). Final BBMV were suspended at a concentration of 16 mg protein/ml in mannitol-Hepes-Tris (MHT) buffer (100 mmol/l mannitol, 20 mmol/l Hepes- Tris, pH 7.4, and protease inhibitor: Aprotinin 20 µg/ml, leupeptin 20 µg/ml, pepsta tin 5 µg/ml, and chymostatin 5 µg/ml). The purity of the preparation was routinely determined by assaying the marker enzyme alkaline phosphatase (E.C.3.1.3.1), which was enriched 8 to 10 fold. BBMV were stored at -70 °C until use and diluted to the concentration of 5 mg protein/ml with MHT buffer for transport studies. 3 .10.2. Transport measurements Uptake of radioactive substrate was determined at 22°C with the rapid filtration technique as described initially by Hopfer (Hopfer et al ., 1973). The uptake was in itiated by adding 20 µl BBMV suspension (100 µg protei n) to 50 µl incubation media to give 100 mmol/l mannitol, 20 mmol/l Hepes-Tris (pH 7.4), 100 mmol/l NaSC N or KSCN, 0.1 mmol/l substrate 5µCi D- [ 3 H] glucose, and the indicated inhibitor concen trations. The incubation was terminated after 5 s by adding 1 ml ice-cold stop solution (100 mmol/l NaCl, 100 mmol/l mannitol, 20 mmol/l Hepes-Tris and 0.2 mmol/l phlorizin, pH 7.4) followed by rapid filtration of the suspension through prewetted 0.45 µm nitrocellulose filters . After rinsing with 3.5 ml ice-cold stop solution the wet filters were dissolved in 7 ml scintillation cocktail and counted for radioactivity in a liquid scintillation counter. Substrate uptakes presented are mean values ± S.E from at least three independent experiments, each performed in duplicate or triplicate. For statistical comparisons, Student’s t- test was used. 3 .10.3. Photoaffinity labeling of trunc ated loop 13 protein with TIPDG or BzG The photolabeling experiments were carried out in 200 µl PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO 4, 1.8 mM KH 2PO 4, pH 7.4) containing 200 µg truncated loop 13 Materials and methods 46 protein and 1 mM TIPDG or with 0.5 mM Bz G. The mixture was preincubated at room temperature in the dark for 5 min. After inc ubation, photolysis was carried out in a Rayonet photochemical reactor RPR-100, f itted with 16, 3500Å lamps at 22 °C for 5 min for TIPDG and for BzG an Oriel photochemical reactor, fitte d with 200 W medium pressure Hg lamp at 25°C for 30 min. As a control, the photoprobe was first exposed to UV light and then added to the protein. No labeling was observed in the absence of UV light. We also performed photoaffinity labeling of truncated loop 13 with 1 mM TIPDG in the presence of 100 µM arbutin and with 0.5 mM BzG in the presence of 15 µM phlorizin under the same conditions that were employed in the other photolabeling experiments. After photoaffinity labeling the protein was precipitated with chloroform methanol (v/v 2:1). The protein pellet was solublized with 0.1% TFA in 50% acetonitrile for MALDI-TOF mass spectrometry analysis. Mass spectra were acquired in the positive ion, linear mode on a Voyager DE-PRO MALDI (PE- Biosystems, Shelton, USA). After mixing the solublized protein with sinapinic acid matrix (saturated sinapinic acid solution in 0.1% TFA in 50% acetonitrile), samples were deposited on a MALDI plate and dried at room temperature. 3 .10.4. Photoaffinity labeling of truncated loop 13 protein with 3-AP Photoaffinity labeling was performed with the photolabile phlorizin analogue 3- Azidophlorizin. The photolabeling experiments were carried out in 200 µl PBS buffer (pH 7.3) containing 200 µg truncated loop 13 prot ein and 1 mM 3-Azidophlorizin. The mixture was preincubated at room temperature in the dark for 5 min. After incubation, photolysis was carried out in a Rayonet photochemical reactor RPR-100, fitted with sixteen, 2800Å lamps at 22° C for 10 min. As a control, the photoprobe wa s first exposed to UV light and then added to the protein. No labeling was observed in the absence of UV light. After photoaffinity labeling the protein was precipitated with chloroform methanol (v/v 2:1). A small part of the protein pellet was solublized with 0.1% TFA in 50% acetonitrile for MALDI-TOF mass spectrometry analysis and the rest of the protein pellet was dissolved in sample buffer for SDS-PAGE. Proteins were separated by SDS- PAGE using NuPAGE 4-12% Bis-Tris gels (Invitrogen). Gels were stained with Cooma ssie Brilliant Blue G-250 (Neuhoff et al., 1985). Bands of interest were excised from gel with a clean razor blade, sliced into 1 mm 3 cubes and incubated over night at room temperature with 200 µl of 50 mM NH 4HCO 3 , pH 8.5, 50% acetonitrile, then dehydrated with 200 µl acetonitrile and dried in a centrifugal evaporator. Gel pieces were rehydrated with a small volume of digestion solution (50 mM NH 4HCO 3 , 5 mM CaCl2, and 12.5 ng/µl of trypsin) at 4 ° C for 30 min. Trypsin was used at an enzyme: substrate ratio of ~ 1:100 by weight. Digestion was allowed to proceed overnight at 37 ° C. Peptides were extracted from the gel pieces with 100 µl of 20 mM NH 4HCO 3 followed by 2 extractions of water: acetonitrile (1:1) plus 1% trifluoroace tic acid and finally, 1 extraction with 100 µl of acetonitrile. All extracts were combined in a fres h tube, flash frozen and dried in a centrifugal Materials and methods 47 evaporator. Dried extracts were stored at -20 ° C until analyzed. Mass spectra were acquired in the positive ion, linear mode on a Voyager DE-P RO MALDI (PE-Biosystems, Shelton, USA). After mixing the solublized protein with 2, 5-dihydroxybenzoic acid matrix (saturated DHB solution in 0.1% TFA in 50% acetonitrile) for intact truncated loop 13 protein, and for acquisition of mass spectrometric peptide map of the trypsin digested protein, 0.5 µl aliquots of the generated cleavage products were dispensed onto the sample support, followed by 0.5 µl of CHCA matrix solution (solution of α-Cyno-4-hydroxycinnamic acid in 0.1% TFA in 50% acetonitrile). Samples were deposited on a MALDI plate and dried at room temperature prior to collecting the spectra. Results 48 4. Results 4.1. E .coli as an expression host 4.1.1. Construction of different cl ones of hSGLT1 in pET22b vector The full-length cDNA sequence of human sodium/ D-glucose cotransporter 1 (hSGLT1) was amplified by PCR. Agarose gel electrophoresis s hows the amplification of 2.0 Kbp fragments (Figure 4.1A) as expected, which corresponds to the gene size of hSGLT1 . In order to further ensure that amplified gene product is hSGLT1 gene we performed its digestion with BamH1 because hSGLT1 gene contains only one BamH1 restriction site. Therefore, after BamH1 digestion it would reveal 2 DNA fragments if the PCR amplified product was correct. The predicted size of 2 DNA fragments is 1576 and 419 bp (which is in total 1995 bp), respectively. Analysis of the digestion mixtur e revealed that 2 DNA fragments were precisely produced by BamHI1digestion (Figure 4.1B). The cDNA sequence encoding full-length hSGLT1 was cloned into pET22b E. coli expression vector by using EcoR1 and Xho1 restriction enzymes sites. The plasmid was transformed into E. coli DH5α strain for further replication. The restriction digestion of this clone with EcoR1 and Xho1 obtained the evidence for the successful construction of pET22b- hSGLT1 plasmid. Figure 4.1 Different steps for the construction of pET22b-hSGLT1 clone. ( A ) PCR amplification of hSGLT1 gene by using EcoR1 and Xho1 restriction sites containing primers from EST clones. Only EST4 clone contains full-length hSGLT1 gene. (B) Agarose gel of BamH1 digested hSGLT1 gene re sulted in 2 DNA fragments of 1576 and 419 bp (total 1995 bp). In addition to the evidence mentioned above, the construct was also sequenced by using appropriate sequencing primer. The full sequence of the construct revealed that the cloning Results 49 process was successful. Other constructs of hSGLT1 in pET22b expression vector were made with help of the same strategy by using their respective set of primers and restriction enzymes. Some-times it is difficult to express full-length tr ansporter but the expression of small parts as functional domains of the transporters can be favored by using suitable hosts. Domains are known as the regions along a single polypeptide chain that can act as independent units. To that extent, they can be excised from the chain, still be shown to fold correctly, and often exhibit biological activity (Kachalsky et al., 1995; Li de La Sierra et al., 2000). So we tried to express the last seven domain of hSGLT (with 11 amino acids N-terminal part) in pET22b expression vector. Sequence encoding ∆N12-377hSGLT1 was amplified by QuikChangeSite-Directed Mutagenesis method by using ∆N12-377SGLT sense and ∆N12- 377SGLT antisense primers. pET22b- ∆N12-377 hSGLT1 clone was confirmed by DNA sequencing. The same clones as mentioned in th e pET22b series were also prepared using the pBAD24 vector. 4 .1.2. Construction of pET22b-Unc -F and pET22b-Unc-F-hSGLT1 clones Unc-F gene encodes the subunit β of F0 membrane sector of E. coli ATP synthase (Walker et al., 1982). Fusion of Unc-F gene at the N-terminal of gene of interest resulted in a polycistronic gene, which encodes two different proteins. Over- expression of β -subunit of the E. coli ATP synthase induces a massive proliferation of membranes in E. coli C43 (DE3) strain, which helps in the proper insertion of membrane proteins (Arechaga et al., 2000), sometimes this means help in the expression of eukaryotic membrane proteins. To test this idea for the expression of hSGLT1 prot ein, we constructed pET22b-Unc-F ( control ) and pET22b-Unc-F-hSGLT1 clones by the ligation of Xba1 digested purified Unc-F gene fragment (511 bp) with similarly treated dephophorylated pET22b and pET22b-hSGLT1 expression vectors. The resulting expression plasmids pET22b-Unc-F and pET22b-Unc-F- hSGLT1 were transformed into DH5 α cells for replication and for DNA isolation. It is a single enzyme ligation so the Unc-F gene can ligate into pET22b and pET22b-hSGLT1 vector into two ways, right orientation or reverse orientation. For the identification of clones containing Unc-F gene in the right orientation we performed colony PCR of these clones with primers: forward T7 promoter and reverse UncF . Clones with right orientation of Unc-F gene only shows amplification of Unc-F fragment (Figure 4.2). Results 50 Figure 4.2 Agarose gel of PCR amplified Unc-F gene product from the clones pET22b-Unc-F and pET22b- Unc-F-hSGLT1 for the identification of positive clones with right orientation of Unc-F gene. Lane PU1 and SU1 indicate the clone containing Unc-F gene in correct orientation. 4 .1.3. hSGLT1 expression in E .coli Since E. coli is the first choice for heterologous expression of any foreign gene, heterologous expression of hSGLT1 was tried in E. coli. For the expression of hSGLT1 each clone was transformed into following E. coli strains (Rosetta, C41, C43, and C43Rosetta) except pET22b-Unc-F-hSGLT1 clone, which was transfor med, into C43 and C43Rosetta cells. For the analytical purpose each clone was grown in 10 ml culture volume in different expression conditions (mentioned in the section 3.6.1 of Material and Methods). When O.D 600 of culture reached between 0.4-0.6, protein expression wa s induced by IPTG (in case of pET22b vector) and by L - (+) arabinose (pBAD24 vector). 1 ml cultu re was withdrawn before and after 4 h of protein induction and protein expression was probed by Western blot analysis by using anti- His tag, anti-SGLT1 and anti-FLAG tag antibodies but none of the clones tested positive for hSGLT1 expression (Table 4.1). Table 4.1 Expression of hSGLT1 in different clones of E. coli Clone E. coli strain Outcome pET22b-hSGLT1 R, C41, C43 and C43R* Expression not observed pET22b-hSGLT1-Spacer Same Same pET22b- ∆N12-377 hSGLT1 Same Same pET22b- ∆N12-377 hSGLT1-Spacer Same Same pET22b-Unc-F-hSGLT1 C43 and C43R Same pET22b-Unc-F-hSGLT1-Spacer Same Same pET22b-Unc-F- ∆N12-377 hSGLT1 Same Same pET22b-hSGLT1-FLAG-Spacer R, C41, C43 and C43R Same pBAD24-hSGLT1-FLAG-Spacer R, C41, C43 and C43R Same pBAD24- ∆N12-377 hSGLT1- FLAG-Spacer R, C41, C43 and C43R Same Results 51 R, Rosetta; C41, C41 (DE3); C43, C43 (D E3); C43R, C43 (DE3) Rosetta.Conditions for expression are described in Material and Methods. However in all Western blot probed by anti- His tag antibody a specific band around 72 kDa (expected molecular weight region of hSGLT1) app ears but this band is also present in control as well as in uninduced samples. The rationale for preparing the various plasmids is briefly summarised in the following paragraph. First we constructed full-lengt h hSGLT1 clone in pET22b vector (pET22b- hSGLT1 series), in which the sequence encoding His tag was just after C-terminal of hSGLT1 gene but in this case we could not see any expression of hSGLT1. Thus we were thinking about highly charged His tag might interfere with the proper insertion of newly synthesized protein into the membrane, which would result in degradation of protein. In order to test this possibility we constructed a second series of clones in pET22b vector (pET22b-hSGLT1- spacer series), in which we added a 10 ami no acids long spacer between hSGLT1 gene and the His tag, but also in this case protein expression results were negative. Because bacterial cotransporter posses significantly shorter N-terminal hydrophilic extensions than those in eukaryotes (Turk and Wright , 1997), we deleted the first seven helices (pET22b- ∆N12-377hSGLT1 series) but also in vain. Taking into consideration a possible advantage associated with co-expression of outer membrane protein Unc-F with our gene of interest, we constructed a new series of expression plasmid in pET22b vector (pET22b-Unc- F-hSGLT1). Results of hSGLT1 expression in this type of clones were also negative. 4 .1.4. β-subunit of E.coli ATPase expression in pET22b-Unc-F and pET22b- Unc-F-hSGLT1 plasmids We tested pET22b-Unc-F and pET22b-Unc-F- hSGLT1 plasmids for the expression of β - subunit of E. coli ATPase in C43 and C43Rosetta strains (Unc-F is toxic for other strains of E. coli ). In this case we could not detect expre ssion of hSGLT1 but SDS-PAGE analysis of uninduced and induced samples from pET22b- Unc-F and pET22b-Unc-F-hSGLT1 indicates quite interesting and unique band pattern (Fi gure 4.3). pET22b-Unc-F clone indicates very good expression of the β -subunit of E. coli ATPase around 20 kDa in the induced sample ( lane 3) while as expected there was no protein band detected in the uninduced sample ( lane 2 ) at this molecular weight. To our surprise this protein band was completely absent in case of pET22b-Unc-F-hSGLT1 clone, uninduced ( lane 4) and induced ( lane 5). The most interesting thing with these results is that we constructed a polycistronic gene in which protein translation is independent from each other but in one case we are getting expression of β - subunit of E. coli ATPase but when we fused the Un c-F gene with hSGLT1 gene the expression of β -subunit of E. coli ATPase was completely abolished. These results lets assume that mRNA of hSGLT1 might be unstable in E. coli and that therefore when expressed simultaneously also the mRNA of the Unc-F gene is destroyed. Results 52 Figure 4.3 Coomassie- stained SDS-PAGE gel for the expression of β- subunit of E. coli ATPase in pET22b-Unc-F and pET22b-Unc-F-hSGLT1 plasmids. M, marker protein; PU0, pET22b-Unc-F uninduced; PU1, pET22b-Unc-F induced by 0.5 mM IPTG after 4 h of induction (position of arro w is indicating expression of β - subunit of E. coli ATPase around 20 kDa); SU0, pET22b-Unc-F-hSGLT1 uninduced; SU1, pET22b-Unc- F-hSGLT1 induce by 0.5 mM IPTG after 4 h of induction. 4 .1.5. Messenger RNA stability of hSGLT1 in E .coli In order to further shed light on mRNA stability of hSGLT1 and its possible implication in heterologous expression of hSGLT1 in E. coli, we performed computational analysis of hSGLT1 mRNA by RNAdraw V1.1B program, wh ich predicts secondary structure and minimum energy (Figure 4.4). Calculated s econdary structure of hSGLT1 mRNA shows energy of –541.99 kCal at 37 °C with 323 CG pairs, 225 AU pairs and 96 GU pairs. Computational analysis revealed that hSGLT1 mRNA sequence contains 5 potential RNase E sites at nucleotide sequence 576, 600, 1488, 1500, and 1558 (Table 4.2). RNase E is an endoribonuclease, which degrades mRNA from 5´- to 3´ direction (Mackie, 1998). RNase E is known to cleave mRNA substrates in single stra nded regions that are proceeded or followed by a stable stem-loop structure (Carpousis et al., 1994; Lin-Chao and Cohen, 1991; Melefors and von Gabain, 1988). Moreover, the stem-loop structures adjacent to the cleavages site do not affect the specificity of cleavages but can influence the rate of cleavage indirectly (McDowall et al., 1995). While originally suggested that an RNase E cleavages site encompasses a 10 nucleotide region (ACAGA/UAUUUG) (Tomcsanyi and Apiri on, 1985), subsequent analysis of many more sites indicate that RNase E prefers single-stranded regions that are typically, but not always A-U rich (Coburn and Mackie, 1999). Add itionally, it is known that RNase E is a 5´- Results 53 end-dependent endoribonuclease (Mackie, 1998) that prefer substrates with monophosphorylated 5´ ends to triphosphorylated termini (Lin-Chao and Cohen, 1991). The predicted RNase E sites in the mRNA of hS GLT1 are thus at positions 1488, 1500, and 1558 (see Table 4.2) and the discussion section. Table 4.2 Predicted RNase E sites in the mRNA of hSGLT1 Cleavage site Nucleotide sequence Stem-loop structure adjacent to cleavage site 576 ACAAUUACAG Absent 600 GUGAUUUACA Absent 1488 GGGAUUUCAC Present 1500 UAUGAUUACU Present 1558 GAUUAUCUGU Present Figure 4.4 Secondary structure of hSGLT1 mRNA as calculated by RNAdraw V1.1B program. Results 54 4.2. Pichia pastoris as an expression host 4.2.1. Construction of expression plasmid pPICZB-hSGLT1 hSGLT1 gene was PCR amplified using an EST clone (DKFZp686N20230Q2) as a template. The cDNA coding for the human sodium/glucose cotransporter hSGLT1 was cloned into the plasmid pPICZB that uses the AOX promoter, which is able to drive over-expression of the hSGLT1 gene in P. pastoris. hSGLT1 was cloned into pPICZB vector under ´ AOX´ promoter. EcoR1 and NotI restriction sites were introduced upstream and downstream of the hSGLT1 gene by PCR and the EcoR1 and Not1 containing gene fragment was cloned into the respective sites of the pPICZB vector (Figure 4.5). To facilitate immunological detection and protein purification, the FLAG epitope and 6His tag were attached to the C-terminus of the hSGLT1. In order to resolve the potential eff ect of 6 His tag on the proper targeting of hSGLT1 in the membrane we placed a 10 am ino acids long spacer (VLYKSGGSPG) between the His tag and the hSGLT1 gene sequence. In the resulting expression vector pPICZB- hSGLT1 the hSGLT1 gene was under the transcriptional control of the AOX1 promoter, which is inducible by methanol (Figure 4.5). For further propagation and growth this plasmid was transformed into TOP10F - E. coli strain and five colonies were grown in 10 ml low salt LB with 100 µg/ml Zeocin and 10 µg/ml Tetracycline for 12 h at 37 °C for plasmid isolation, which was used for the identification of positive clones by EcoR1 and Not1 restriction digestion (Figure 4.6) and by DNA sequencing. Figure 4.5 hSGLT1 expression construct. cDNA sequence encoding full-length hSGLT1 cloned into pPICZB vector in EcoR1 and NotI restriction sites in the multiple cloning site (MCS), for the expression of hSGLT1. Figure 4.6 Eco R1 and Not 1 restriction digestion for the identification of positive colony of pPICZB- hSGLT1 clone. Lane pPICZB E/N, pPICZB vector digested by EcoRI and NotI restriction enzymes; Lane1-5, colonies of pPICZB-hSGLT1 clone digested by EcoR1 and Not1 restriction enzymes. Only positive colonies (1, 2, 3, and 5) yield fragments of 2000 bp after EcoR1 and Not1 restriction digestion. MCS hSGLIT-FLAG-Spacer-His MCS EcoR1 Not1 A OX 1 pPICZB Results 55 4.2. 2 . Identification of positive clones of hSGLT1 showing protein expression Recombinant derivates of plasmid pPICZB linearized by digestion with Pme1 typically integrate at the AOX locus as a single copy, but multiple copy integration occurs with a frequency of 1 to 10%. Resistance to higher le vels of Zeocin correlates with higher copy number for the integrated plasmid, which usually (Romanos et al., 1998) but not always (Clare et al., 1991) correlates with higher levels of expression of the recombinant protein of interest. In our case clones selected on 1000 µg/ml Zeocin concentration give the highest yield of hSGLT1 as judged by Western blot analys is by anti-FLAG antibody (Figure 4.7), so for further expression we used the clone of this plate. Figure 4.7 Western blot analysis by anti-FLAG antibody for the identification of positive clones for hSGLT1 expression. Clones 1 and 2 were selected from 500 µg/ml Zeocin plate whereas 3 and 4 were selected from 1000 µg/ml Zeocin plate. Clones 1, 2, 3, and 4 show expression of hSGLT1. Clones from 1000 µg/ml Zeocin plate shows highest expression of hSGLT1. 4 . 2 . 3 . Optimization of feeding and induction in the shake flask cultures For expression of proteins using the AOX1 promoter it is important to keep the methanol level within a relatively narrow range. In bioreactors this can be achieved by different control methods (Cereghino and Cregg, 2000). For expressi on in shake flasks Guarna (Guarna et al., 1997) introduced a technique for on-line monitoring of methanol concentration. This still requires significant technical effort. Our aim wa s to empirically optimize a feeding protocol for our host strain (GS115, methanol using genotype), which works without any measuring devices and is, therefore, suitable for optimiza tion. Sofar most expression protocols for shake flask scale are based on the recommendations by Invitrogen Corporation, USA, where 0.5% methanol (v/v) are added to start induction a nd the rest addition of 0.5% (v/v) methanol occurs after 24 h. To determine the optimum course of methanol feeding we varied the amount of methanol added to the culture. We decided to add methanol twice a day instead of 24 h to minimize concentration shifts in the medium. The medium used was BMMY. Our results shows that after 24 h of induction a methanol feed of 1% twice a day led to a 2 times higher protein expression than a methanol feed of 0.5% (v/v ) twice a day as judged by Western blot by anti- Results 56 FLAG antibody (Figure 4.8). Sofar induction with 1% (v/v) methanol and feeding with 1% (v/v) methanol twice a day gave optimal results in our expression system. Figure 4.8 Western blot analysis by anti-FLAG antibody for the optimization of feeding and induction in shake flasks. Clones from 1000 µg/ml Zeocin plate were grown in the same condition but induced in different conditions. 1, 2, 3 induced and feed with 1% methanol tw ice in 24 h. 4, 5, 6 same clones (1, 2, 3) induced and feed with 0.5% methanol once in 24 h. 4 . 2 . 4 . Purification of hSGLT1 from Pichia pastoris The clone with the highest protein yield was grown in BMGY medium supplemented by 100 µg/ml Zeocin for 30-36 h at 28 °C for O.D 600 of 2-4/ml and then transferred to methanol containing medium (BMMY with 100 µg/ml Zeocin) to induce protein expression for 24 h. After expression, cells were disrupted in break ing buffer containing protease inhibitor cocktail with vigorous vortexing with acid washed chilled 0.5 mm glass beads. The cell pellet obtained by high-speed ultra centrifugation was washed w ith 4 M urea to remove peripheral membrane proteins. The stripped membranes were solublized in 1.2% FosCholine-12 in TG buffer at 4 °C overnight and the solublized material was removed by high-speed centrifugation. The clear supernatant was applied to a preequilibrated Pr otino Ni-resin column for the purification of hSGLT1 by using buffer TG with 250 mM imidazole. The obtained protein was more than 95% pure as judged from Coomassie stained SDS ge ls (Figure 4.9A). Western blot analysis of different steps of hSGLT1 purifi cation by anti-FLAG antibody (Figure 4.9B, lane I, L1, P1, and S4 ) reveal some proteolytic degradation of the recombinant transporter (protein bands around 30 kDa range), even though all purifi cation steps were carried out at 0-4 °C. These degradation products were completely absent in purified hSGLT1 (Figure 4.9B, lane hSGLT1 ). Purified hSGLT1 migrates on SDS-PAGE at an apparent mass of 55 kDa (Figure 4.9A, lane hSGLT1 ), which corresponds to that of the nonglyc osylated transporter (Firnges et al., 2001; Panayotova-Heiermann et al., 1997; Quick and Wright, 2002) and is recognized by anti- FLAG antibody (Figure 4.9B) and by anti-hSGLT1 and anti-His tag antibodies. The 1-liter culture of P. pastoris gives 3 mg of purified protein. Results 57 Figure 4.9 Coomassie- stained SDS-PAGE gel and immunoblot of it with anti-FLAG antibody during different stages of hSGLT1 purification. (A) M, protein marker; UI, uninduced sample; I, induced sample after induction of 1% methanol; L, whole cell lysate after lysis of Pichia pastoris cells expressing hSGLT1; S1, supernatant; P1, pellet after high-speed centrifugation; UW, washing obtained after 4 M urea wash to pellet P1; S4, supernatant obtained after solublization of urea wash pellet in 1.2% FosCholine-12; and hSGLT1, Protino column purified hSGLT1 which shows an apparent molecular mass of 55 kDa (marked by arrow). (B) Western blot of above mentioned SDS-PAGE gel with anti-FLAG antibody. 4 . 2 . 5 . Functional analysis of recombinant hSGLT1 in right-side-out membrane vesicles Functionality of hSGLT1 was confirmed by determining sugar uptake (50 µM α-MDG) by right-side-out vesicles of P. pastoris GS115 producing hSGLT1, shown in Figure 4.10, when stimulated by sodium a rate of 273 ± 11 nmol × mg of membrane protein-1 × min-1 is observed. The uptake is almost completely blocked by phlorizin (29 ± 4 nmol × mg of membrane protein-1 × min-1 ). For the control vesicles, there was no significant difference in sugar uptake in the presence or absence of Na+ (33 ± 3 nmol × mg of membrane protein-1 × min-1 ), transport was also not affected by the addition of phlorizin. Results 58 0 5 0 100 150 2 0 0 2 5 0 3 0 0 Control Sodium Sodium + Phlz Potassium α -MD G Up ta ke (n m ol /m g of m em br an e pr ot ei n x m in ) Figure 4.10 Sugar uptake by right-side-out vesicles of P. pastoris expressing hSGLT1. 50 µM α-MDG uptake by right-side-out vesicles of P. pastoris GS115 producing hSGLT1 is stimulated by sodium (Na + , 100 mM) and inhibited by phlorizin (Phlz, 10 µM). No sodium-dependent, phlorizin-sensitive uptakes were observed with vesicles of GS115 harboring plasmid pPICZB as a c ontrol. For the control vesicles, there was no significant difference in sugar uptake in the presence or absence of Na+ (33 ± 3 nmol × mg of membrane protein-1 × min-1 ), transport was also not affected by the addition of phlorizin. 4 . 2 . 6 . Functional analysis of recombinant hSGLT1 in prteoliposomes For reconstitution of purified hSGLT1, performed liposomes made of 90% (w/v) asolectin soy lecithin and 10% (w/v) cholesterol were titr ated with Triton X-100 and solublization was followed by turbidity measurements. The detergent concentration was adjusted to the onset or total solublization of lipid. After mixing of the detergent-destabilized liposomes with the purified transporter, detergent was removed by addition of Bio-Beads SM-2 in three steps, first two steps at room temperature and third step at 4°C overnight. The time course of 10 µM α-MDG uptake into the proteoliposomes in the presence and absence 100 mM Na+ , and in the presence of 100 µM phlorizin is shown in Figure 4.11. In the presence of 100 mM Na+ , sugar transport peaked within 30 min at 313 ± 13 nmol α-MDG per mg of recombinant hSGLT1 before falling to the concentration equilibrium of about 60 nmol α-MDG per mg of recombinant hSGLT1 in more than 5 h time. The time course of α-MDG uptake in proteoliposomes demonstrated a classical overshoot (Kinne, 1976; Lucke et al., 1978) in the presence of Na + , indicating that sugar was concentrated within the Results 59 proteoliposomes and that uptake was energised by the Na+ electrochemical gradient via secondary active cotransport. The overshoot is absent if there is no sodium gradient across the membrane. These finding indicate a coupling of sodium flux to glucose flux. 0 5 0 100 150 200 250 3 0 0 3 5 0 0 100 200 300 Time (min) α − MD G Up ta ke (n m ol /m g of h SG L T 1) 100mM Sodium 100microMole Phlorizin Control 100mM Potassium Figure 4.11 Time course of sugar uptake into prot eoliposomes containing purified recombinant hSGLT1. The time course of 10 µM α-MDG in the presence of 100 mM sodium ( — ) shows a 35 fold of increase in the initial rate of sugar uptake before reaching concentration equilibrium in 5 h. After addition of 100 µM phlorizin (  ) or removal of Na + ( W ), the initial rate was identical to control liposomes ( • ) and sugar accumulation above the equilibrium was not observed. In order to further check the functionality of purified recombinant hSGLT1, we performed sugar uptake in the proteoliposomes composed of hSGLT1 asolectin soy lecithin and cholesterol. Proteoliposomes take-up 1874 ± 65 nmol × mg-1 hSGLT1 × min-1 (Figure 4.12) as compared to 125 ± 24 nmol × mg-1 hSGLT1 × min-1 by liposomes alone. Sodium-dependent α-MDG uptake was completely blocked by 10 µM phlorizin (Figure 4.12). Phlorizin is a potent high-affinity competitive i nhibitor of sugar transport by SGLT1 in native tissue, brush border membrane vesicles, Xenopus oocytes, and reconstituted in proteoliposomes (Diedrich, 1966; Diez-Sampedro et al., 2001; Ikeda et al., 1989; Quick and Wright, 2002; Schultz and Curran, 1970) with a half-maximum inhibition constant ( KiPhlorizin ) in the micromoler range. In our study, 10 µM phlorizin inhibited the initial rate of α-MDG transport in membrane vesicles by 89% (Figure 4.10), whereas the same concentration of phlorizin in proteoliposomes inhibits uptake by 86% (Figure 4.12). Results 60 0 5 0 0 10 0 0 15 0 0 2 0 0 0 2 5 0 0 Control Sodium Sodium + Phlz Potassium α -MD G Up ta ke (n m ol /m g of h SG L T 1 x m in ) Figure 4.12 Sugar uptake kinetics of proteolip osomes containing purified recombinant hSGLT1. 50 µM α- MDG uptake by proteoliposomes made of purified hSGLT1 transport 1874 ± 65 nmol × mg of hSGLT1 -1 × min-1 in a sodium-dependent manner (when 100 mM Na + was replaced by 100 mM K+ transport slows down to 598 ± 3 nmol × mg of hSGLT1 -1 × min-1 ) and 10 µM phlorizin inhibited sugar transport upto 251 ± 39 nmol × mg of hSGLT1 -1 × min-1 ), whereas liposomes (control) transported only 125 ± 24 nmol × mg of hSGLT1 -1 × min-1 . The sugar selectivity of recombinant hSGLT1 in proteoliposomes was measured by determining the uptake of 100 µM α-MDG in the presence of 10 mM D-Glucose ( D-Glc), D- Galactose ( D-Gal), 2-Deoxy- D-Glucose (2Doglc), Mannose (Man), Allose (All), L - Glucose ( L -Glc), and 100 µM phlorizin (Phlz) in the presence of 100 mM Na + . High-affinity substrates of hSGLT1 D-Glc and D-Gal inhibited Na + -dependent α-MDG by 70% and 72%, while low affinity substrates 2Doglc, Man, All, and L -Glc inhibited sugar uptake 44%, 18%, 15%, and 2% respectively and in the presence of 100 µM phlorizin the Na + -dependent sugar uptake was reduced to 84% of original value (Figure 4.13, Table 4.3). hSGLT1 in proteoliposomes exhibits substrate specificity in the following order, Phlz >> α-MDG ≈ D-Glc ≈ D-Gal >> 2Doglc > Man > All > L -Glc. Recombinant reconstituted hSGLT1 shows sterospecificity as it preferred D-Glu over L -Glu, D-Glu inhibited 75% α-MDG uptake L -Glu, inhibited only 2%. Furthermore, the sugar selectivity of the reconstituted recombinant hSGLT1 appears to be similar to that reported for transporter in different systems (Diez-Samp edro et al., 2000; Firnges et al., 2001; Kinne, 1976; Quick and Wright, 2002). The equatorial orientation of the OH-group at the positions C-2 and C-3 of the D-glucose seems to be important for its recognition by hSGLT1 since in Results 61 the presence of mannose, 2-Deoxy- D-glucose and allose the significant inhibitory effect on α- MDG uptake was observed. The importance of the equatorial OH-group orientation at the C-2 and C-3 was also reported for the renal SGLT1 (Burckhardt and Kinne, 1992). The 78% inhibition of α-MDG uptake in the presence of D-galactose indicates that the orientation of OH-group at C-4 (equatorial in D-glucose and axial in D-galactose) is not that important for the recognition by cotransporter because D-Glu and D-Gal inhibited sugar uptake by transporter upto equal extent (Table 4.3), Phlz inhibited 84% of α-MDG uptake in proteoliposomes. 0 2 0 40 6 0 8 0 100 120 None D-Glc D-Gal Phlz 2-doGlc L-Glc Man All % α-MDG Uptake Figure 4.13 Sugar uptake kinetics of proteolipos omes containing purified recombinant hSGLT1. Uptake of 100 µM α-MDG is inhibited by high-affinity substrates or inhibitors of hSGLT1 (10 mM D-glucose, D-Glc; 10 mM D-galactose, D-Gal; 100 µM phlorizin, Phlz) but not by low-affinity substrates (e.g., 10 mM 2-deoxy-D- glucose, 2Doglc; 10 mM mannos e, Man; 10 mM allose, All; 10 mM L- glucose, L-Glc). Table 4.3 Effect of various substrates and inhibitors on the uptake of 100 µM α-MDG as shown in Figure 4.13. Substrate/ Inhibitor Concentration used % of α-MDG uptake inhibition None 100 µM 0 D- Glu 10 mM 75 D- Gal 10 mM 78 2- Doglc 10 mM 44 Man 10 mM 18 All 10 mM 15 L- Glu 10 mM 2 Phlz 100 µM 84 Results 62 For the determination of sugar uptake kinetics parameters, 1 min α-MDG uptake assays were performed by varying α-MDG concentration from 0.01 to 25 mM. A half–saturation constant (K m sugar) of 0.4 ± 0.05 mM/L and a maximum velocity (Vmax) of 3.4 ± 0.34 µmol × mg hSGLT1 -1 × min-1 was observed (Figure 4.14). The carrier catalytic turnover number (King et al., 1993; Napoli et al., 1995), an index of num ber of sugar molecules transported per transporter was calculated by dividing the sugar transport Vmax by the total number of transporter present in the proteoliposomes. Catalytic turnover of hSGLT1 based on the Vmax of 3.4 µmol × mg hSGLT1 -1 × min-1 is 6 s -1 (Table 4.4), which is in good agreement with that obtained in previous studies (Quick and Wright, 2002) and other Na + -dependent transporter [e.g., the Na + /glucose transporter of Vibrio parahaemolyticus (vSGLT) (Turk et al., 2000), and the Na+ / proline transporter of Escherichia coli (PutP) (Jung et al., 1998)]. 0 5 0 0 1000 1500 20 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0 4 5 0 0 0 5 10 15 20 25 [ α-MDG] (mM) α -MD G Up ta ke (n m ol /m g of h SG L T 1x m in Figure 4.14 Sugar uptake kinetics of proteolip osomes containing purified recombinant hSGLT1. Concentration dependence of the initial-rate sugar uptake shows a half-saturation constant ((K m sugar) of 0.4 ± 0.05 mM/L and maximum velocity (Vmax) of 3.4 ± 0.34 µmol × mg hSGLT1 -1 × min-1 . Table 4.4 Kinetics of hSGLT1 in different systems. Xenopus oocytes ∗ Proteoliposomes• Proteoliposomes This study Kinetic parameters hSGLT1 hSGLT1 ∆N-GFP hSGLT1 Km sugar, mM/L 0.41 ± 0.03 0.38 ± 0.1 0.4 ± 0.05 Turnover number, s-1 50 4 6 •Reported values in previous studies(Quick and Wright, 2002). Results 63 4.3. Synthesis and Characterization of different photoaffinity probes In order to study the functional domains of SGLT1 photoaffinity labels were synthesized. They were derived from phlorizin a non-trans ported inhibitor, from arbutin, a transported inhibitor of SGLT1 and a D-glucose based label. The aim of these studies is to obtain information about the important contact points within the transporter at different depth and in various conformations. In the following section we describe the synthesis, photochemical properties, stability, effects of these probes on SGLT1 mediated glucose upt ake in natural membranes and photoaffinity labeling of an isolated recombinant functional domain of SGLT1. In the future above labels will be employed for the photoaffinity labeling of whole recombinant hSGLT1. 4 . 3 .1. Synthesis of labels BzG and 3-AP for the identification of phlorizin interaction sites Figure 4.15 Chemical structure of photoa ffinity probes and their sibling. (A) Phlorizin highest-affinity inhibitor of sugar uptake by SGLT1, (B) [ (4´-Benzoyl) phenoxy]-D- glucopyranoside (BzG), (C) 3 - A zidophlorizin (3-AP). BzG and 3-AP were synthesized to identify phlorizin interactions sites in the cotransporter. The scheme for BzG synthesis (compound 12) is given in Figure 3.3. Compound 11 was obtained by the reaction of α- D-acetobromoglucose 4 with 4-hydroxybenzophenone 10 in the presence of mercuric cyanide, which was further deacetylated to give final compound 12 in 98% yield. The synthesis scheme for 3-AP (compound 16) was shown in Figure 3.4. 3-Nitrophlorizin (compound 14) was obtained by simple nitrati on of phlorizin, which was subsequently A B C Results 64 reduced to 3-aminophlorizin (compound 15). The transformation of 3-aminophlorizin into 3- azidophlorizin by reacting it in the cold with dilute hydrochloric acid and sodium nitrite followed by addition of sodium azide to the diazonated phlorizin represents a classical reaction in organic chemistry. The infrared absorption at 2170 cm -1 of the final product is characteristics for the azido group. 4 . 3 . 2 . Synthesis of label TIPDG for th e identification of arbutin ineraction sites In order to get information about arbutin binding sites in cotransporter we have synthesized TIPDG. The reaction scheme for the synthesis of TIPDG 9 is presented in Figure 3.2. The coupling of α- D-acetobromoglucose 4 with 4-hydroxy-3- iodo-trifluoroacetophenone 3 gave the corresponding keto compound 5, in 75% yield. Keto compound 5 on reaction with hydroxylamine hydrochloride gave oxime 6, which was finally converted into aceto TIPDG 8 by the reaction of oxime 6 with triethylamine, DMAP, P-toluenesulfonyl chloride, and NH 3 and oxidation of diaziridine 7 with iodine. TIPDG 9 was obtained after deacetylation of compound 8. Figure 4.16 Chemical structure of photoaffinity probe and its sibling. (A) Arbutin, a high-affinity sibling of TIPDG transported by SGLT1, (B) [(2’-Iodo -4’- ( 3 ’ ’ -trifluoromethyldiazirinyl) phenoxy]-D -glucopyranoside (TIPDG). 4 . 3 . 3 . Synthesis of label 6-AG for the identification of D-glucose interaction sites D-Glucose is a high-affinity transported substr ate of SGLT1, in order to identify its binding sites in cotransporter we have synthesized 6-AG photoprobe. The synthesis of 6-AG (compound 22) is depicted in Figure 3.5. The di rect conversion of the debenzylated aldehyde 17 into the diazirine as described (Lehmann and Thieme, 1986) gave only unsatisfactory results. Therefore the aldehyde 17 was converted into the corresponding methyl ketone 19 by reacting with methylmagnesium iodide followed by oxidation of the formed secondary alcohol. The less-reactive keto compound 19 coul d be easily converted into the diazirine A B Results 65 group with the NH3 , Hydroxylamine-O-sulfonic acid reagen t and subsequent oxidation of the intermediate diaziridine with iodine (Church et al., 1972; Church and Weiss, 1970). Figure 4.17 Chemical structure of photoaffinity probe and its sibling. (A) D-glucose transported ligand by SGLT1, (B) n-Methyl-6C-(Azimethyl)-D- glucopyranoside (6-AG). 4 . 3 . 4 . Photochemical properties and st ability of TIPDG, BzG, 3-AP and 6- AG Consistent with the spectral properties of other 3-(trifluoromethyl)-3-phenyldiazirine (Brunner et al., 1980; Delfino et al., 1993; Fang et al., 1998; Hatanaka et al., 1994; Nassal, 1984; Platz et al., 1991), the TIPDG described here shows a characteristic absorption maximum at 358 nm ( ε = 290 M -1 .cm-1 ). This is depicted in Figure 4.18 for TIPDG 9 photolysis studies. When a 0.5 mM solution of 9 in methanol was irradi ated in a Rayonet photochemical reactor RPR-100 fitted with 16, 3500Å lamps at 22 °C, the diazirine absorption ba nd at 358 nm decreased with a half-life of 20.1 s following apparent first-orde r kinetics. Upon photolysis, the solution turned slightly orange (absorbance maximum at around 460 nm), a result of the well documented partial rearrangement of the diazirine to the diazo isomer. There have been reports on the reduction of photoactivable reagents containing azido groups (Bayley and Knowles, 1978) or 2-diazo-3, 3, 3-trifluoropropionyloxy groups (Takagaki et al., 1983) by commonly used reducing agents like dith iothreitol, 2-mercaptoethanol and reduced glutathione. To check the stability of TIPDG in the presence of these reducing agents, a methanolic solution of TIPDG was treated with 2- mercaptoethanol and dithiothreitol at an equimolar concentration or in the presence of a 5-fold excess of these reagents at 25 °C. The reaction was followed spectrophotometrically wherein no change was observed up to 1 h. The diazirine moiety of TIPDG is also stable under hydrogenation with 10% Pd on carbon, which leads to a non-iodinated TIPDG derivative. A B Results 66 Figure 4.18 UV spectra of the reaction pr oducts from photolysis of TIPDG 9 . A solution (0.5 mM) in methanol was irradiated for different times (0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 s). The spectra show the decay of the characteristic diazirine absorption with a maximum at 358 nm . The half-life of decomposition was calculated from a logarithmic plot of the absorbance data against the irradiation time. The half-life t1/2 was 20.1 s. BzG shows absorption maximum at 293 nm ( ε = 22500 M -1 .cm-1 ) and reveals other characteristic features of benzophenone phot ochemistry (Dorman and Prestwich, 1994; Dorman and Prestwich, 2000). The half-life of photolysis of BzG was determined by irradiating a 0.5 mM solution in isopropanol in an Oriel photochemical reactor fitted with 200 W medium pressure Hg lamp at 25°C. The characteristic absorption band of benzophenone in BzG at 293 nm disappeared with an approximate half-life of 25.4 min following first order reaction kinetics. 3 - AP exhibits photochemical properties similar to other aryl azide compounds. The absorption maxima of azide group of 3-AP are at 211 nm ( ε = 28150 M -1 .cm-1 ), 258 nm ( ε = 6700 M - 1.cm-1 ), and 288 nm ( ε = 16800 M -1 .cm-1 ). Half-life of decompos ition of this compound was not determined. The absorption maximum of the diazirine group of 6-AG is at 330 nm ( ε = 45 M -1 .cm-1 ). On irradiation with long wavelength UV light ( λmax around 350 nm) in a Rayonet photochemical reactor, the 6-AG decompose with a half-life of 1.3 min. 4 . 3 . 5 . Inhibition of glucose uptake in rabbit small intestine BBMV by BzG and 3-AP To determine the affinity of BzG towards the SG LT1 cotransporter we measured its inhibitory potential on sodium-dependent D-glucose uptake into rabbit intestinal BBMV according to the method of Dixon. The effect of BzG on Na + -dependent glucose uptake in small intestine Results 67 BBMV is shown in Figure 4.19. BzG clearly acts as a fully competitive inhibitor. The apparent Ki value 12 ± 2 µM is only slightly higher than the previously reported Ki value of 8 ± 1.2 µM (Evers et al., 1978; Gibbs et al., 1982) phlorizin. Figure 4.19 Inhibitory effects of 0, 10, 20, 30, 40, and 50 µM BzG on 5 s sodium-dependent D-glucose uptake into rabbit small intestine BBMV measured at two subs trate concentrations, 0.1 mM (squares) and 0.2 mM (circles) D-glucose according to the method of Dixon. Representative experiments are shown. An inhibition constant ( Ki) of 12 ± 2 µM was calculated from three independent experiments. The affinity of 3-AP for the SGLT1 cotran sporter was measured according to method of Dixon. 3-AP strictly act as a high a ffinity inhibitor of sugar uptake by Na+ / D-glucose cotransporter with a Ki value of 8.6 ± 1.6 µM. The calculated Ki value for 3-AP is exactly similar to its parent compound phlorizin ( Ki of 8 ± 1.2 µM). 3-AP exhibits all characteristics features similar to phlorizin so we used th is compound for the identification of phlorizin- binding sites in truncated loop 13 protein which is predicted to be region involved in inhibitor binding (Novakova et al., 2001; Xia et al., 2003). 4 . 3 . 6 . Inhibition of glucose uptake in rabbit small intestine BBMV by TIPDG As shown in Figure 4.20 TIPDG exhibits a fu lly competitive inhibition with respect to D- glucose with a Ki of 22 ± 5µM, this affinity is almost identical to the one of its parent compound arbutin (25 ± 6 µM). The modification of the phenyl ring OH group into a trifluoromethyl diazirine group and the mono iodination of its phenyl ring thus did not significantly alter the affinity to the transporter. Results 68 Figure 4.20 Inhibitory effects of 0, 25, 50, 75, and 100 µM TIPDG on 5 s sodium-dependent D-glucose uptake into rabbit small intestine BBMV measured at two subs trate concentrations, 0.1 mM (squares) and 0.2 mM (circles) D-glucose according to th e method of Dixon. Representative experiments are shown. From three independent experiments a Ki value of 22 ± 5 µM for TIPDG was calculated. 4 . 3 . 7 . Inhibition of glucose uptake in rabbit small intestine BBMV by 6-AG The affinity of 6-AG for the SGLT1 cotrans porter was also evaluated by measuring its inhibitory potential on sodium-dependent D-glucose uptake into rabbit small intestine BBMV according to method of Dixon. As shown in Fi gure 4.21, 6-AG exhibits a fully competitive inhibition characteristic with respect to D-glucose with a Ki of 40 ± 5 µM. Compared to the inhibitory effect of D-glucose ( Ki of 1 mM) the modification of the molecule with the photolabile diazirine group and methyl group at position C-6 of the D-glucose moiety drastically increase the affinity for the transporter. These results indicate that presence of alkyl and diazirine group (hydrophobic interaction) in crease the affinity of 6-AG 25 fold as compared to its parent compound. Results 69 Figure 4.21 Inhibitory effect of 0, 100, 150, and 200 µM 6-AG on 5-s D-glucose uptake into rabbit small intestine BBMV measured at two subs trate concentrations, 0.1 mM (squares) and 0.2 mM (circles) D-glucose according to the method of Dixon. Representative experiments are shown. From three independent experiments Ki value of 40 ± 5 µM for 6-AG was calculated. Table 4.5 Photochemical and biochemical pr operties of different photoaffinity probes. Photoaffinity probe Sibling λmax nm ( ε = M -1 .cm-1 ) Half-life of photolysis (t 1/2 ) Inhibition constant ( Ki ) photoaffinity probe Inhibition constant ( Ki ) of sibling TIPDG Arbutin S 358(290) a 20.1 s 22 ± 5 µM 25 ± 6 µM BzG Phlorizin I 293(22500) b 25.4 min 12 ± 2 µM 8 ± 1.2 µM 3-AP Phlorizin 211(28150) c 258(6700) c 288(16800) c NDd 8.6 ± 1.6 µM 8 ± 1.2 µM 6-AG D-Glucose S 330 (45 ) a 1.3 min 40 ± 5 µM 1 mM S Substrate I Inhibitor a Methanol b Isopropanol c Ethanol d not determined Results 70 4.4. Photoaffinity labeling of trunc ated loop 13 with TIPDG, BzG, and 3-AP According to studies employing site-direct ed mutagenesis (Novakova et al., 2001) and molecular recognition atomic force microscopy (Wielert-Badt et al., 2002), the extra- membranous loop 13 of SGLT1 appears to be i nvolved in substrate and inhibitor recognition of SGLT1 (Panayotova-Heiermann et al., 1997; Panayotova-Heiermann et al., 1996; Xia et al., 2003). We therefore used this functional domain of SGLT1 (a model system loop 13 in its truncated form that has amino acids sequen ce 564-638 of SGLT1) for th e identification of substrates and inhibitor sites. 4 . 4 .1. Photoaffinity labeling of truncated loop 13 with TIPDG and BzG Figure 4.22 MALDI-mass spectrometry of photoaffinity labeled truncated loop 13. (A) Truncated loop 13 before labeling, (B) Photoaffinity labeling with TIPDG and (C) Photoaffinity labeling with BzG. Truncated loop 13 protein (200 µg) was incubated with 1 mM TIPDG or with 0.5 mM BzG in 200 µl of PBS buffer for 5 min in the dark at room temperature followed by irradiation at 350 nm for 5 min (TIPDG) and 30 min (BzG). After separation from noncovalently bound TIPDG or BzG by chlo roform/methanol extraction, protein was dissolved in SA matrix at a concentration of 50 pmol/µl and subjected to MALDI- mass sp ectrometry, (A) peak I, truncated loop 13 protein; (B) peak I, truncated loop 13 protein; peak II, truncated loop 13 protein cross-linked with TIPDG; (C) peak I, truncated loop 13 protein; peak II, truncated loop 13 protein labeled with BzG. A B C Results 71 Photoincorporation of TIPDG into truncated loop 13 proteins was complete within 1 min of light exposure under our condition. Figure 4.22A shows the expected mass peak at m/z 8825.6 Figure 4.22B demonstrates that after photoaffinity labeling with TIPDG two peaks of m/z 8 825.6 and 9287.5 can be observed. Peak I corres ponds to truncated loop 13 protein and peak II to the photolabeled truncated loop 13 protei n (9287.5). The mass difference between peak I and peak II (Figure 4.22B) corresponds to the mass of photolysed TIPDG (461.9). MALDI-mass spectra of photolabeled loop 13 prot eins with BzG are shown in Figure 4.22C. Two intense ion signals at m/z 8825.6 and at m/z 9187.2 are observed. The ion signal at m/z 9187.2 was absent in the MALDI-mass spectrum of control truncated loop 13 protein Figure 4.22A. The mass increment of 361.6 amu sugge sts a simple carbon bond insertion of the photoaffinity label BzG into the protein (Figure 4.22C, peak II ). 4 . 4 . 2 . Photoaffinity labeling of truncated loop 13 with 3- Azidophlorizin The reaction product of photolabeling of the truncated loop 13 with 3-azidophlorizin was also analysed by MALDI-mass spectrometry. Figure 4. 23A shows the expected mass peak for loop 13 at m/z 8825.60 and in Figure 4.23B the produc t after photoaffinity labeling of the loop with 3-azidophlorizin is depicted. Peak I corre sponds to truncated loop 13 (8825.60) and peak II to the photolabeled truncated loop 13 protei n (9275.10). The mass difference between peak I and peak II (Figure 4.23B) corresponds to the mass of photolysed 3-azidophlorizin (449.5). Figure 4.23 MALDI-mass spectrometry of photoaffinity labeled truncated loop 13. Protein (200 µg) was incubated with 1 mM 3-azidophlorizin in 200 µl of PBS buffer for 5 min in the dark at room temperature followed by irradiation at 280 nm for 10 min. Afte r separation from noncovalently bound 3-azidophlorizin by chloroform/ methanol extraction, protein was dissolved in DHB matrix at a concentration of 50 pmol/µl and subjected to MALDI-mass spectrometry, (A) Control experiment; peak I, truncated loop 13, (B) Products after photolabeling; peak I, truncated loop 13; peak II, truncated loop 13 cross-linked with 3-azidophlorizin. A B Results 72 4.4. 3 . Identification of the ligand contact points As a next step in identification of the liga nd contact points, the photolabeled-truncated loop 13 was digested in gel with trypsin. After extrac tion of the peptides from the gel, the resulting mixture was analyzed by MALDI-TOF mode with unlabeled-truncated loop 13 as a reference (Table 4.6). The only additional peak observed in the labeled probe was at m/z 624.52 (Figure 4.24B). This can be explained as an adduct peak to m/z 175.11 (corresponding to Arg-602) with a difference of 449.41. The latter is equal to the mass of photolysed 3-azidophlorizin. All other peaks appear also in the control as shown in Figure 4.24A. Figure 4.24 MALDI-mass spectrum of in-gel trypsin cleaved truncated loop 13. 200 µg of truncated loop 13 was photolabeled with 3-azidophlorizin, proteins were se parated on SDS-PAGE and protein bands of interest were excised, destained and digested in gel with trypsin. The peptides were extracted from the gel pieces with acetonitrile. Subsequently 0.5 µl ali quots of the generated cleavage products were dispensed onto the sample support, followed by 0.5 µl of CHCA matrix solution and the MALDI- mass spectra were recorded. (A) Truncated loop 13 before photoaffinity labeling, (B) truncated loop 13 after photoaffinity labeling with 3- azidophlorizin. (*) peak indicates the mass shift due to modification of Arg-602 with 3-azidophlorizin. A B Results 73 Table 4.6 Theoretical and measured masses of photolabeled truncated loop 13 digested with trypsin as shown in Figure 4.24. Fragment Amino acids Sequence Theoretical mass (M+H) + monoisotopic Measured mass (M+H) + Additional peaks 1 1-3 PER 401.21 ND a 2 4-6 NSK 348.18 ND 3 7-9 EER 433.20 ND 4 10-33 IDLDAGEEDIQEAPEEATDTEVPK 2614.18 2614.37 5 34 K 147.11 147.27 6 35 K 147.11 147.27 7 36 K 147.11 147.27 8 37-40 GFFR 526.27 526.11 9 41 R (602) 175.11 175.35 624.52 b 10 42-53 AYDLFCGLDQDK 1387.61 1387.63 11 54-56 GPK 301.18 ND 12 57-59 MTK 379.20 380.85 13 60-66 EEEAAMK 807.35 807.26 14 67-68 LK 260.19 ND 15 69-77 LTDTSEHPL 1012.49 ND a not determined. b Denominates a peptide plus label We also performed photoaffinity labeling of truncated loop 13 with 1 mM 3-AP in the presence of 15 µM phlorizin under the same conditions that were employed in the other photolabeling experiments. MALDI- mass spectru m of photolabeled truncated loop 13 did not show any additional peak in intact truncated loop 13 and also no additional peak was present in the Trypsin digested samples. In order to further strengthen our results that labeling of residue Arg (602) is specific and it is not an artifact, we chemically modified the guanidino moiety of arginine with phenylglyoxal treatment. Photoaffinity labeling of arginine-modi fied protein with 3-AP did not resulting any additional peak in the MALDI- mass spectrum of trypsin digested photolabeled protein. Taking these evidences into consideration we can claim that the ortho position of ring B of phlorizin labeled amino acid Arg-602 of truncat ed loop 13, which thus forms part of a phlorizin-binding site. Discussion 74 5. Discussion Integral membrane proteins such as channels and transporters are essential for all living organisms as they mediate the passage of ions and molecules across the membrane. The understanding of how membrane proteins work in health and disease has been restricted hitherto largely due to lack of purified protein. Most membrane proteins are present in very low concentrations in their native tissue, and their over-expression is a prerequisite for purification. The use of eukaryotic expression system has illuminated mechanistic features of human membrane proteins, but these are unsuitable for their large-scale purification (Fleming, 2000; Tate, 2001). In addition, genetic disorders of membrane proteins are frequently caused by improper trafficking in cells, and it is not known whether missorted proteins retain their catalytic activity. The first crystal structures of membrane proteins were obtained with proteins isolated from naturally abundant sources, and recombinant proteins still represent minority of the membrane proteins that have been crystallized to date. In the case of soluble proteins, over 90% have been crystallized using recombinant protein, whic h is in contrast to the 21 recombinant of 50 total structures for membrane proteins (Loll, 2003). Only three mammalian membrane proteins to date have had crystal structures determined using recombinant protein: prostaglandin H2 synthase-2 (COX-2), produced in insect cells (Barnett et al., 1994; Kurumbail et al., 1996), monoamine-oxidase B, produced in yeast (Binda et al., 2002) and fatty acid amide hydrolase, produced in bacteria (Bracey et al., 2002). All three proteins are monotopic membrane proteins and therefore have little hydrophobic surface. It should also be noted that the only GPCR crystallized to date , bovine rhodopsin, was obtained from natural source, from bovine retina where it is found in large amounts. These statistics reflect the fact that active recombinant membrane proteins are difficult to obtain. It is not surprising that heterologous production of membrane proteins poses big challenges. Unlike their cytoplasmic counterparts, membrane proteins have a longer way to the fully functional form. Once their synthesis begins, th e complicated secretory machinery is engaged in order to achieve proper targeting and insertion into the membrane. The majority of proteins synthesized in many cells are cytoplasmic protei ns; such cells have no sufficient capacity to handle large amounts of newly synthesized membra ne proteins. Therefore, when produced in a heterologous system, membrane proteins can “saturate” the secretory pathway of cell, leading to protein aggregation inside different cell compartments. Therefore, the choice of expression system is a crucial step towards obtaining large amounts of transporter in its active form. Devising a successful strategy for its subsequent purification is nonetheless important. For the expression of hSGLT1, various animal heterologous expression systems have been widely used but major disadvantages associated with these systems are that modified protein are frequently not trafficked properly to plasma membrane Discussion 75 ( Lam et al., 1999; Lostao et al., 1995; Martin et al., 1997; Martin et al., 1996) and that it is costly to produce sufficient amounts of protein for structural studies. Recently functional expression of hSGLT1 ∆N-GFP was reported in E. coli ( Quick and Wright, 2002), but hSGLT1 expression in E. coli was not reproducible in our lab as reported in the results section. 5.1. E . coli as an expression host Among the many systems available for heterol ogous protein production, the Gram-negative bacterium E. coli remains one of the most attractive because of its ability to grow rapidly and at high density on inexpensive substrates, its well-characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains. Taking advantages of E. coli expression system into consideration, first we attempted expression of hSGLT1 in this system. For the expression of hSGLT1, we used two types of vectors, pET22b (T7 promoter based) and pBAD24 (P BAD promoter), both having some advantages and disadvantages. We constructed different clones of hSGLT1 in pET22b and pBAD24 and tested hSGLT1 expression in different E. coli strains and at different conditions (Table 4.1) but we were not able to express hSGLT1 in E. coli. 5 .1.1. Probable reasons for not getting hSGLT1 expression in E. coli There are some probable reasons for not getting expression of hSGLT1 in E. coli. 5.1.1.1. Codon usage E. coli and indeed all cells, use a specific subset of the 61 available amino acid codons for the production of most mRNA (Zhang et al., 1991). So-called major codons are those, which occur, in highly expressed genes, whereas the minor or rare codons tend to be in genes expressed at low level. The hSGLT1 gene wa s directly amplified from human genome and cloned into different expression vectors in E. coli, therefore, the codons of this gene are not optimized for E. coli expression host. Codon degeneracy analysis shows that the hSGLT1 gene contains 23% codons, which cannot be transl ated efficiently, due to the low frequency of the corresponding tRNAs in E. coli. Therefore, a special host strain Rosetta (DE3) was used for the expression of hSGLT1 in prokaryote. Rosetta host strains are BL21 derivatives, designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNA genes for AGG (Arg), AGA (Arg), AUA (Val), CUA (Leu), CCC (Pro), GGA (Gly) on a Col-E1 compatible chloroamphenicol-resistant plasmid. Thus, the Rosetta strains provide for “universal ” translation, which is otherwise limited by the codon usage of E. coli. Transcription of the tRNAs genes is driven by their native promoters. The use of this strain did not solve the problem of expression. Thus, codon usage does not Discussion 76 appear to be a hurdle for the expression of hSGLT1. This signifies the importance of some other factors, which are responsible for the expression of human membrane proteins in E. coli. 5 .1.1.2. Signal sequences Signal sequences or signal peptides are a we ll-characterized but heterogeneous group of cleavable N-terminal peptide extensions that di rect the newly synthesized proteins to specific subcellular compartments. In E. coli ~20 aa signal peptides direct secretion of proteins to the periplasma and outer membrane (Arkowitz a nd Bassilana, 1994; Driessen, 1994; Driessen et al., 2001). In eukaryotes, signal peptides are more variable in length and direct secretion to the endoplasmic reticulum lumen and the general secretory pathway (Sakaguchi, 1997). There are however, distinctions between the prokaryotic and eukaryotic systems; most notable are differences in the amino acid sequences of the signal peptide itself (Nielsen et al., 1997). In addition there are differences in the process of secretion: i.e., the function of the signal recognition particle (SRP), a more highly evolved secretion apparatus, and the use of different chaperones (Pohlschroder et al., 1997). If E. coli signal sequences were not working for hSGLT1 protein we should get hSGLT1 expression e ither in the form of aggregated protein in inclusion bodies, or in degraded forms, but our results indicate that this was not the case since we could not detect any sign of protein expression in the E. coli expression system. We also attempted expression of hSGLT1 at different conditions by changing the protein inducer and temperature from 37 to 16 °C to change folding and degradation, but in all cases no expression was observed. Therefore in our experiments it seems that some phenomena other than those mentioned above could be the real cause for not getting expression of hSGLT1 in E. coli. 5.1.1.3. Degradation of hSGLT1 mRNA in E .coli As already shown by the results obt ained by SDS-PAGE analysis of β - subunit of E. coli ATPase expression in pET22b-Unc-F and pET22b- Unc-F-hSGLT1 plasmids (Figure 4.3), the expression of this protein was completely abolished when simultaneous expression of both the β -subunit and hSGLT1 was attempted. Subseque nt computational analysis of the mRNA sequence of hSGLT1 revealed that it contains 5 potential RNase E sites at position 576, 600, 1488, 1500, and 1558 (Table 4.2). The position 576 (ACAAUUACAG) and 600 (GUGAUUUACA) contains the prefe rred nucleotide sequence (Figure 5.1) for degradation by RNase E but they are lacking a stem-loop stru cture adjacent to the cleavage site. RNase E prefers AU rich nucleotide sequences with ad jacent secondary structure (stem-loop) for degradation. So these two sites may not be all that prone for RNase E degradation. The three predicted RNase E sites at position 1488 (GGGAUUUCAC), 1500 (UAUGAUUACU), and 1558 (GAUUAUCUGU) (Table 4.2) contain extended stem loop structures adjacent to these sites (Figure 5.2), so these sites are highly su sceptible for degradation by RNase E. Once the RNase E degradation process is initiated in E. coli, it activates other exonucleases which Discussion 77 further degrade mRNA of the Unc-F gene. Thus the lack of hSGLT1 expression may be due to the instability of the mRNA. The role of cyclic nucleotide regulation of Na+ /glucose cotransporter (SGLT1) mRNA stability for the expression in LLC-PK1 cells was reported in the literature (Lee et al., 2000), same observations for the effect of mRNA st ability on glucose transporter (GLUT1) gene expression were also mentioned in previous studies in mammalian cell lines (Qi and Pekala, 1999). Figure 5.1 Part of hSGLT1 mRNA secondary structu re indicating the positions of RNase E sites. Nucleotide sequences in this structure marked as 576, I (ACAAUUACAG); 600, II (GUGAUUUACA); 1488, III (GGGAUUUCAC); and 1500, IV ( GAUUAUCUGU) indicates the positions of RN ase E susceptible site in the mRNA of hSGLT1. To our knowledge, this is the first time that possible implications of mRNA stability of hSGLT1 on its expression into E. coli are reported. The reason given by most of membrane protein molecular biologists to explain their unsuccessful attempts is that the membrane protein is synthesized in E. coli but due to the absence of specialized membrane protein folding machinery (Molecular Chaperones) a nd proper membrane targeting machinery in bacteria, expressed protein either accumulates as insoluble aggregates of improperly folded species called inclusion bodies, or proteolytic degradation of newly synthesized protein takes place due to improper targeting of protein. In our opinion, this statement does not hold true in I II III IV Discussion 78 all conditions and the major culprit of our failure for hSGLT1 expression in E. coli is the mRNA stability of hSGLT1. Figure 5.2 Part of hSGLT1 mRNA secondary structu re indicating the position of RNase E site. Nucleotide sequence 1558 of hSGLT1 mRNA marked as V (GAUUAUCUGU) in this structure is highly prone for RNase E degradation due to presence of very high stem-loop structure adjacent to this site. According to our mRNA degradation hypothesis, cDNA of hSGLT1 is transcribed into mRNA of hSGLT1 without any problem. At this point as mentioned above since hSGLT1 mRNA contains 5 RNase E sites, RNase E a nd other endoribonucleases start to degrade hSGLT1 mRNA from the 5´-3 ´ end. RNase E de gradation also activates other exonucleases like PNPase, RNase II, and RNase R. These active exonucleases start mRNA degradation from 3´-5´ end of oligoribonucleotides generate d by RNase E degradation into smaller parts. These smaller parts are further degraded into mononucleotides by oligoribonucleases. 5 .1.1.4. Comparison to other attempts to express hSGLT1 in E . coli In the year 2002, Quick and Wright (Quick and Wright, 2002) reported functional expression of hSGLT1 in E. coli. The authors of this study claimed that they obtained 1 mg functional hSGLT1 protein from 3-liters of bacterial culture. These results are only apparently contradictory to the results we obtained in E. coli expression host in the present study. We Discussion 79 analysed their plasmid construction strategy and results in a step by step manner to find out the probable reasons for their success. They used pTMH-6FH expression vector (Quick and Jung, 1998), a derivative of pT7-5 ( lacY) (Bibi and Kaback, 1990) and it works under the lac promoter/operator (Bibi and Kaback, 1990; Quic k and Jung, 1998), which is a mild promoter. Use of mild promoter does not seem to be real reason for their success because we also used pBAD expression vector, which works on the mild promoter P BAD of the araBAD operon. The second important thing in their methodology was the use of growing temperatures below 20°C and the use of BL21 E. coli strains but we employed same growth conditions with same and different E. coli strains and could not get expression of hSGLT1 even in a small amount. The most interesting and important aspect of their study is that, they shortened the N-terminal of hSGLT1 by 16 amino acids (s equence of hSGLT1 from 12-28 aa ), this deletion increases the amount of hSGLT1 inserted into the membrane of E. coli by about fivefold. They reported that bacterial cotransporters possess significantly shorter N-terminal hydrophobic extensions than those in eukaryotes (Turk and Wright , 1997) so this deletion makes hSGLT1 more similar to its bacterial counterpart and its helps in hSGLT1 proper insertion into membrane. Another reason behind the fivefold increment in hSGLT1 expression and insertion into the membrane might, however, be stability of ∆N12-28hSGLT1 mRNA. Deletion of 48 nucleotide sequences at the N-terminal of hSG LT1 resulted in a secondary structure, which contains stem-loops at the N-terminal of ge ne (Figure 5.3). These N-terminal stem-loop structures prevent or delay the mRNA degrada tion by RNase E. Recently with the help of stem-loop addition at 5´end of mRNA, increased expression of genes has been described, which are either not possible to express (Paulu s et al., 2004) or express at very low-level (Carrier et al., 1998; Carrier and Keasling, 1997a; Carrier and Keasling, 1997b; Carrier and Keasling, 1999; Smolke et al., 2000; Smolke and Keasling, 2002) in E. coli. Another important point of their strategy is the addition of glycophorin A transmembrane span (as a 15th transmembrane domain in hSGLT1 sequen ce) and of the green fluorescence protein (GFP) at the 3´end of hSGLT1 gene. It is a well-documented fact that GFP mRNA does not contain any RNase E sites (Smolke and Keasli ng, 2002). Presence of stem-loop structure at the 5´of ∆N12-28hSGLT1-Gly-GFP gene, protects it from RNase E degradation and due to the addition of GFP at the 3´of gene, provides protection to the gene from the exoribonuclease degradation, which acts from 3´-5´end. There are again evidences that indicate that mRNA stability was the decisive factor. Discussion 80 Figure 5.3 Secondary structure of a part of hSGLT1 mRNA, and ∆N12-28 hSGLT1 mRNA. (A) mRNA of hSGLT1 does not contains any stem-loop structure at its N-terminal so it is highly susceptible for RNase E degradation. (B) Deletion of amino acids from N-terminal of hSGLT1 results in a secondary structure, which contains stem-loop at N-terminal of gene, and these stem-loop structure protects the RNase E sites from RNase E degradation. 5.2. P. pastoris as an expression host The methylotropic yeast P. pastoris is a powerful tool for the heterologous expression of protein (Cereghino and Cregg, 2000). This sy stem combines several advantages of prokaryotic and eukaryotic expression systems. The techniques needed for molecular genetic manipulation are similar to those well established for S. cerevisiae. P. pastoris can be easily grown to high cell densities using defined minimal media and is able to introduce eukaryotic posttranslational modifications. Another advantage of the P. pastoris expression system for structural analysis is the possibility of 13 C labeling of expressed proteins for nuclear magnetic resonance analysis. Feeding 13 C labeled methanol provides a cost-effective possibility to achieve incorporation levels of up to 98%. 5 . 2 .1. Expression of hSGLT1 in P. pastoris For the functional expression of hSGLT1 in P. pastoris FLAG tag was introduced in hSGLT1 by changing the native peptide sequence D 574 AEEEN to D 574 YKDDDDK and a 10 amino acids long spacer was placed at C-terminal. Add ition of spacer at C-terminal helps in the proper targeting of hSGLT1 in the membrane becau se if the His tag is directly present at the C-terminal of hSGLT1 it interferes with the insertion of proteins into the membrane (Turk et al., 2000) after its synthesis in the cytosol and we observed degradation of most of the protein under this condition. P. pastoris GS115 harboring a plasmid with hSGLT1 cDNA under the control of the AOX promoter proved to be most suitable for the high-level expression and stability of recombinant hSGLT1. The transporte r was detected in the yeast cells by using a A B Discussion 81 FLAG antibody against the engineered FLAG epitope (Figure 4.9B), and it is active as judged by sodium-stimulated sugar ( D-glucose) uptake. 5 . 2 . 2 . Protein purification For the purification of hSGLT1 we adopted a new strategy, which includes washing of hSGLT1 containing membranes with 4 M urea to get rid of most of peripheral and outer membrane proteins, which helps in the enrichment of hSGLT1. As judged by Western blot analysis during different stages of hSGLT1 pur ification, the hSGLT1 was properly inserted into the membrane because in the supernatant (Figure 4.9B, lane S1 ) and in urea washing (Figure 4.9B, lane UW ), we could not detect any hSGLT1 protein, whereas all hSGLT1 was present in the pellet (Figure 4.9B, lane P1 ), and could be released into supernatant (Figure 4.9B, lane S4 ), which was obtained after solublization of membranes in 1.2% FosCholine-12. A variety of molecular signals or mechanisms have been reported for the specific localization of particular membrane proteins to specific membrane compartments (Bibi et al., 1992; Matter et al., 1992; Sandoval and Bakke, 1994; Weingla ss and Kaback, 2000) but the mechanism by which hSGLT1 inserts into the membrane is still unclear. Recently, Quick and Wright (Quick and Wright, 2002) showed that shorter N-term inal of hSGLT1 increases the amount of hSGLT1 inserted into the E. coli membrane because bacterial cotransporters posses significantly shorter N-terminal hydrophilic extens ions than those in eukaryotes (Turk and Wright, 1997). However in P. pastoris most of hSGLT1 inserted into membrane without any modification at its N-terminal as judged by Wester n blot analysis (Figure 4.9B). These results indicate that signal sequences encoded within hSGLT1 sequence are fully functional in the P. pastoris. Purified hSGLT1 shows an apparent molecu lar mass of 55 kDa, which corresponds to the nonglycosylated transporter. Despite the apparent nonglycosylated status of hSGLT1 in P. pastoris, its shows biochemical properties that are indistinguishable from glycosylated hSGLT1. This finding also supports previous observations that N- glycosylation is not required for hSGLT1 activity (Quick and Wright, 2002; Turk et al., 1996). Recombinant hSGLT1 was purified by Ni-affinity chromatography on a Protino column to more than 95% purity as judged by Coomassie Blue staining SDS-PAGE gel (Figure 4.9A). No degradation product was observed in the purified hSGLT1 (Figure 4.9A and B). About 3 mg of hSGLT1 was purified from 1 liter of P. pastoris culture. The yield of hSGLT1, which we obtained in this study, was higher than those reported for other membrane protein in P. pastoris expression system. The yield for other membrane protein reported in literature are as following, 2 mg of multidrug resistance protein1 (Cai et al., 2001), 0.7 mg of ATP-binding cassette transporter (Cai and Gros, 2003), 2.2 mg of NOP-1 (Bieszke et al., 1999), and 1 mg of 5HT5A -serotoninergic receptor (Weiss et al., 1998). Discussion 82 5.2. 3 . Reconstitution and functional analysis of recombinant hSGLT1 Purified recombinant hSGLT1 was reconstitu ted into performed detergent-destabilized liposomes made of 90% (w/v) asolectin soy lecithin and 10% (w/v) cholesterol with a detergent concentration adjusted to the onset of solublization (Jung et al., 1998; Panayotova- Heiermann et al., 1999; Rigaud and Levy, 2003) . 100 mM sodium stimulated uptake of 50 µM α-MDG is completely blocked by 10 µM phlorizin (Figure 4.10, Figure 4.11, Figure 4.12, and Figure 4.13). In order to further charact erize recombinant hSGLT1 in proteoliposomes, we performed competitive uptake studies of 100 µM α-MDG in the presence of inhibitor (100 µM phlorizin) high-affinity substrates (10 mM D-glucose and D-galactose) and low-affinity substrates (10 mM 2-Deoxy- D-glucose, mannose, allose, and L -glucose) inhibited sugar uptake according to their affinity towards transporter (Table 4.3). hSGLT1 in proteoliposomes exhibits substrate specificity in the following order, Phlz >> α-MDG ≈ D-Glc ≈ D-Gal >> 2Doglc > Man > All > L -Glc. Catalytic turnover of hSG LT1 based on the Vmax of 3.4 µmol × mg hSGLT1 -1 × min-1 is 6 s -1 (Table 4.4), which is in good agreement with that obtained in previous studies (Quick and Wright, 2002) and other Na + -dependent transporters [e.g., the Na+ / glucose transporter of Vibrio parahaemolyticus (vSGLT) (Turk et al., 2000), and Na + / proline transporter of Escherichia coli (PutP) (Jung et al., 1998)]. The kinetics of recombinant hSGLT1 in intact cells or membrane vesicles , and reconstituted proteoliposomes, mirror those determined for hSGLT1 in native tissue and Xenopus oocytes. 5 . 2 . 4 . Reasons for getting functional expression of hSGLT1 in P. pastoris One obvious question that comes into mind, if mRNA of hSGLT1 is unstable in E. coli and contains five potential RNase E sites why did we get expression of hSGLT1 in P. pastoris. The best possible explanation of this ques tion is that the mRNA half-life and underlying degradation mechanisms are different in P. pastoris as compared to E. coli. In yeast, the degradation of the transcript begins with the shortening of the poly (A) tail at the 3´end of mRNA (Muhlrad and Parker, 1992; Shyu et al ., 1991). Shortening of the poly (A) tail primarily leads to removal of the 5´-cap stru cture (decapping), thereby exposing the transcript to digestion by a 5´-3´exonuclease. In addition to decapping and 5´-3´decay, transcripts can also be degraded in a 3´-5´direction following deadenylation (Anderson and Parker, 1998; Muhlrad et al., 1995). This process is cataly zed by a conserved complex of multiple 3´- 5´exonucleases, termed the exosome that also performs a wide variety of RNA processing events. Currently, the available data suggest that the major mechanism of mRNA decay in yeast is by decapping and 5´-3´degradation (B eelman et al., 1996). Yeast mRNA can also be degraded by mechanisms that involve specific cleavage of transcript independent of prior poly (A) shortening. Discussion 83 Decapping is a key step in the yeast mRNA degrad ation for several reseaons. First, in the 5´- 3´pathway of mRNA degradation, it both precedes and permits the decay of the transcript body. Second, since individual mRNAs are decapped at different rates, decapping is also an important control point in mRNA decay. Third, decapping also plays a critical role in the process of mRNA surveillance wherein aberrant mRNAs containing nonsense codons are very rapidly decapped and degraded in a 5´-3´d irection. The major difference in the mRNA degradation mechanism in E. coli and P. pastoris is that the decapping is the most crucial step in the mRNA degradation in yeast but in bact eria this step is not necessary for mRNA degradation. Average half-life of mRNA in yeas t is around 3 h while in case of bacteria it is only 1.5 min. Second explanation for hSGLT1 expression in P. pastoris can be given on the basis of difference in the codon usages in both expression systems. As mentioned previously, codon usage in E. coli are ~23% bias for hSGLT1 gene as compared to H. sapiens, while codon usage in P. pastoris are only 9.7% bias as compared to H. sapiens. Third possible explanation is that P. pastoris as a eukaryote organism shares many similarities with H. sapiens at the cellular level and molecular mechanisms underlying transcription, translation and membrane trafficking of membrane proteins compared to their prokaryote counterpart E. coli. 5 . 2 . 5 . Advantages of our expression method Our method of expression of hSGLT1 in P. pastoris has certain advantages over other methods of hSGLT1 expression. We expre ssed full-length hSGLT1 protein without any significant modification in hSGLT1 as comp ared to previously reported hSGLT1 in E. coli ( Quick and Wright, 2002), in which N-terminal of hSGLT1 was shortened by deleting amino acid residues 12-28 and GFP was fused to its C-terminal but in our opinion hSGLT1 ∆N-GFP is not suitable for most of the biochemical and biophysical applications due to its very high molecular weight ( ≈100 kDa) and due to intense modifications in it. The amount of hSGLT1 (3 mg hSGLT1 per 1-liter of yeast culture) obtai ned in our expression system is the highest yield reported so far for hSGLT1 prot ein expression. The yield obtained in E. coli is only 1 mg of purified recombinant protein per 3-liter of cultured bacterial cells. For the better immunological detection and to provide ease in the purification two affinity tags are present in our protein. Our protein purification method is very simple and efficient. The initial step involving washing of the protein pellet with 4 M urea resulted in a much more enriched hSGLT1 protein pellet which was further solub lized in 1.2% FosCholine-12. Thus resultant solublized supernatant gave more than 95% pur ified hSGLT1 protein without any degradation product on Ni-affinity purification on a Protino column, while the other reported procedure includes two steps protein purification, immuno-affinity chromatography followed by gel filtration (Quick and Wright, 2002). Advantages of our hSGLT1 expression methods clearly Discussion 84 demonstrate that our method is novel and unique , as it provides purified protein in less time with less expenditure. 5 . 3 . Photoaffinity labeling of truncated loop 13 A photoaffinity probe should satisfy the followi ng criteria for its successful utilization in target site identification. It should be small, so as not to cause unacceptable steric perturbation to the system under examination. It should be stable in the dark, and highly susceptible to light at a wavelength that does not cause photolytic damage to the biological sample. Finally, the reaction with biological targets should lead to stable products to enable their isolation, purification and analysis. Both diazirine a nd benzophenone based photo probes exhibit these major advantages over frequently used azi do photoprobes (Bayley and Knowles, 1978; Gibbs et al., 1982; Lin et al., 1982). TI PDG, 6-AG and BzG come closer to satisfying the chemical and biological criteria required for most photo reagents (Brunner et al., 1980; Delfino et al., 1993; Dorman and Prestwich, 1994; Fang et al ., 1998; Hatanaka et al., 1994; Nassal, 1984; Platz et al., 1991; Prestwich et al., 1997). A ll three photoprobes TIPDG, 6-AG and BzG can be photolysed at 350-360 nm, a voiding protein-damaging wavelengths. In TIPDG the electron withdrawing trifluoromethyl group may lead to an increased stability of the C-I bond. That this type of iodinated phenyldiazirine makes pr omising photolabeling reagents is inferred also from a number of studies with 3-(trifluoromethyl)-3-(3 - [ 125I]iodophenyl) diazirine, a reagents widely used in the past for labeling of the apolar phase of membranes (Brunner, 1989; Brunner and Semenza, 1981; Brunner et al., 1983). Th e superb selectivity of this reagent in labeling integral (versus peripheral) proteins may be a direct reflection of the C-I bond stability during diazirine photolysis. Despite the early concerns about the photochemistry of aliphatic as opposed to aromatic diazirines (Brunner, 1993), they have now been successfully employed in the design of minimally perturbing photoaffinity probes (Kipp et al., 1997; Liessem et al., 1995). We, therefore, recently developed a novel photoreactive aliphatic diazirine-containing photoprobe 6-AG that mimic glucose, a high-affinity substr ate transport by hSGLT1. In contrast to the bipolar azido group, no polarization of the ali phatic diazirine group can be observed. The carbon atom of the three membered aliphatic diazi rine ring has a chemical shift of ~15 ppm in 13 C NMR spectra, and the hydrogen atoms of vicinal methyl group have a chemical shift of ~1 ppm in 1H NMR spectra. This data is typical for the chemical shifts usually observed in nonpolar alkyl groups. The nonpolar character of 6- AG seems to have a special benefit for the binding of modified substrates to hydrophobic membrane proteins. BzG exhibits three distinct chemical and bi ochemical advantages over previously reported azido derivatives of phlorizin (Gibbs et al., 1982; Lin et al., 1982), which gave very inconsistent poor labeling results due to harsh photolysis conditions and low labeling efficiency caused by undesired side reactions. First BzG is more stable than azido derivatives Discussion 85 of phlorizin. Second BzG can be manipulated in ambient light and can be activated at 350-360 nm. Third BzG reacts preferentially with unreativ e C-H bonds, even in the presence of solvent water and bulk nucleophiles. The relative inertness of the BzG triplet state to the water should guarantee that no BzG photoaffinity label will be lo st to reaction with water, in contrast to carbene photoaffinity labels, where immediately or delayed hydrolysis has frequently been observed (Galardy et al., 1974; Hexter and We stheimer, 1971; Shafer et al., 1966). Aryl azides are the most widely used photochemical reagents in the strategy of photoaffinity labeling receptors sites on proteins. Photoactivation of aryl azide yields several reactive intermediates, including singlet (half-life on the order of milliseconds) plus small fractions of the relatively long-lived triplet nitrenes. Speci al attention must, therefore, be given to minimize spurious labeling attendant with reactiv e species that can migrate from the original binding sites. Nitrenes are also thought to react preferentially with nucleophilic groups and rarely are involved in random, near-neighboring hydrogen extractions and insertion into C-H bonds. However, this feature is not necessarily a disadvantage in the employment of the phlorizin aryl azide derivative. For example, the receptor for 3-AP probably includes a polar group that serves as the specific interaction site for the –N 3 moiety. Our rationale for the synthesis of 3-AP photoprobe was based on previous results reported by Diedrich group (Diedrich, 1990; Gibbs et al., 1982; Hosa ng et al., 1981), using 4-azidophlorzin that introduction of the azido group into the phlorizin backbone neither changes its interactions mode with Na+ / D-glucose cotransporter nor its inhibition kinetics. The synthesis of the new photoactivable analogue of arbutin 9 features several noteworthy steps: (a) regioselective introduction of an iodi ne atom into the aromatic ring of compound 1 (Figure 3.2) in the presence of excess HgO affo rding aryl iodide 2 in high yield (Brunner et al., 1980; Hatanaka et al., 1994), (b) O-Demethylation of 2, followed by coupling of phenol 3 , in the presence of mercuric cyanide to give keto compound 5 in very high yield (Ambroise et al., 2000). Regioselective introduction of the iodine atom into the aromatic ring of TIPDG can also serve the purpose of producing a radioactive analogue of TIPDG with a high specific activity by using the tritiodeiodination reaction (Ambroise et al., 2000) To check this possibility, we performed hydrodeiodination of TIPDG in the presence of molecular hydrogen and 10% Pd/C, which gave non iodinated form of TIPDG within 1 h without any significant loss of the diazirine moiety. Ambroise (A mbroise et al., 2000) synthesized [(4’-(3’’ - trifluoromethyldiazirinyl)phenoxy]- D-galactopyranoside by stannic tetrachloride-catalyzed glycosylation of β - D-galactose pentaacetate with [3-(4-hydroxyphenyl)-3-(trifluoromethyl)- 3H-diazirine in 43% yield. The phenol [3-(4-hydroxy phenyl)-3-(trifluoromethyl)-3H- diazirine was synthesized in six steps by the pr eviously reported procedure (Hatanaka et al., 1994), but the major drawback of this synthesis was the low yield (26%) of demethylation of [3-(4-methoxyphenyl)-3-(trifluoromethyl)-3H-diaziri ne with trimethylsilyl iodide and a prolonged reaction time 65 h (Hatan aka et al., 1994). In our method, demethylation of 3-Iodo- 4-methoxy-trifluoroacetophenone 2 with LiCl gives phenol 3 in 88% yield and the coupling Discussion 86 reaction of phenol 3 with α- D-acetobromoglucose 4 in the presence of mercuric cyanide gives [(2’-Iodo-4’-trifluoroacetyl)phenoxy]-2,3,4,6-tetra-O-acetyl- D-glucopyranoside 5 in 75% yield. Thus, our method provides a simple and versatile approach for the synthesis of different sugar based photoaffinity probes. The results on Na+ -dependent glucose uptake show that TIPDG (Figure 4.20), BzG (Figure 4.19), 3-AP, and 6-AG (Figure 4.21) in teract with the high affinity Na+ / D-glucose cotransporter. It was further demonstrated that the Ki values of TIPDG (22 ± 5 µM) and its prototype compound arbutin (25 ± 6 µM), BzG (12 ± 2 µM) and its respective model compound phlorizin (8 ± 1.2 µM), 3-AP (8.6 ± 1.6 µM) and its parent compound phlorizin (8 ± 1.2 µM) and of 6-AG (40 ± 5 µM) and its sibling D-glucose (1 mM) were essentially identical. Modification of phenyl ring OH group in arbutin into a trifluoromethyl diazirine group and mono iodination of its phenyl ring neither altered the affinity to the Na+ / D-glucose cotransporter nor the mode of inhibition. To further test the potential of TIPDG and Bz G as a selective photoaffinity probe for SGLT1, we labeled the truncated loop 13 (amino acid 564-638 of SGLT1 amino acid sequence). MALDI-mass spectrometry analysis of photoaffin ity labelled truncated loop 13 protein with TIPDG and with BzG clearly indi cates that our probes successfully labeled the part of SGLT1 protein. Site-directed mutagenesis studies suggest that a hydrophobic region located in the C-terminal loop 13 (amino acids 604-610) is critically involve d in the binding of phlorizin (Novakova et al., 2001). The binding of phlorizin to loop 13 c ould be confirmed in solution by monitoring phlorizin-dependent fluorescence quenching of the endogenous tryptophan residue (Trp-561). It has been further demonstrated that the phloretin (but not the glucose) moiety of phlorizin interacts with loop13 (Xia et al., 2003). Phlori zin consists of a pyranoside ring and two aromatic rings joined by an alkyl spacer. The c onformation of phlorizin in aqueous solutions was previously studied by two-dimensiona l NMR and pharmacophore analysis by Wielert- Badt (Wielert-Badt et al., 2000). Their model indicates that the interactions via hydrogen bonds from the 2-, 3-, 4-, and 6-hydoxyl groups of the pyranoside and the 4- and 6-OH groups of aromatic ring A are essential for phlorizin binding. This information can now be combined with the Trp fluorescence data (Raja et al., 2003) to estimate the dimensions of the phlorizin- binding and recognition region. We can assume that phlorizin binds to the region very close to position 611 by H-bonding probably with 4- OH group by acceptor/donor atom O-4 of aromatic ring A. Thus, position 611 is supposed to be very important in phlorizin recognition, as also suggested by the lower affinity of D611W mutant. The H-bonding of any one hydrophobic amino acid located in region 606-609 with 6-OH group by acceptor/donor atom O-6 of aromatic ring A of phlorizin also causes a strong interaction. The combination of photoaffinity labeling, SDS-PAGE, enzymatic fragmentation, and MALDI-mass spectrometry analysis with resulting allocation of the attachment site of the ortho position of ring B of phlorizin to amino acid Arg-602 now defines directly the phlorizin Discussion 87 binding site in loop 13. Thus, an interaction of this region with the B ring of phlorizin can be assumed. Thereby the binding pocket could accommodate the phlorizin molecule in the predicted conformation. On the basis of phot oaffinity labeling pattern of truncated loop 13 with 3-azidophlorizin, we could provide a genera lized mechanism by which phlorizin inhibits sugar transport by SGLT1. According to our assumption, a phlorizin-binding region in SGLT1 is located between amino acids 601-611, in this region aromatic rings (A and B) of phlorizin interact with hydrophobic amino acids a nd the glucoside moiety of phlorizin is free to interact with sugar interaction sites of SGLT1 (Lo and Silverman, 1998; Panayotova- Heiermann et al., 1997). In summary, this study provides first example of large-scale functional expression and purification of full-length human Na + / D-glucose cotransporter. Recombinant hSGLT1 retains full functionalities when reconstituted into proteoliposomes. The ability to purify functional cotransporter enables a battery of biochemical and biophysical experiments to probe the structure and function of hSGLT1, with results po ssibly relevant to other cotransporters in the SGLT family. We hope that our method of func tional human membrane protein expression in P. pastoris presented in this study may also provide guidelines for the expression of other medically important human membrane proteins which thus far could not be expressed in prokaryote hosts. 5 . 4 . Future prospective In the life of a membrane protein molecular biologist the happiest day is the day when he/she gets functional expression of target protein. Our success in the functional expression of hSGLT1 open doors of new opportunities to work on hSGLT1. It would be an interesting and challenging task to decipher the molecular basis of the sugar transport and inhibitor binding. What are the responsible amino acids in the protein for sugar transport and inhibitor binding? What would be sugar translocation and inhibitor binding mechanisms? These and many other questions can now be readily answered because now we have an expression system, which provides sufficient amount of functional hSGLT1. Photoaffinity labeling provides a promising approach for the identification of sugar translocation pathway and inhibitor binding sites (Raja et al., 2004; Ra ja et al., 2003; Tyagi and Kinne, 2003), so it would be a challenging and interesting task to performed equilibrium and time-dependent photoaffinity labeling studies with reconstituted recombinant hSGLT1 for the identification of critical amino acids responsible for sugar selectivity, uptake and for inhibitor binding. It would be interesting to address the questions related to sugar translocation pathway and inhibitor binding mechanism by means of luminescence spectroscopy, which has proven informative in the case of some other transporter (Vazquez-Ibar et al., 2003) and which could Discussion 88 ultimately provide interesting insight concerning molecular mechanisms responsible for transport activity. The ultimate goal of protein biologist working on structure-functional relationship of protein is to get the information about target protein structure by X-ray crystallography or Nuclear magnetic resonance spectroscopy, so it would be interesting and challenging tasks to work on this aspect of hSGLT1 structure with the help of protein obtained from our expression system. Summary 89 6. Summary Human sodium/D-glucose cotransporter (hSGLT1) is a member of solute carrier family (SLC5) and plays a central role in the homeost asis of glucose, salt, and water. During the course of this work, first the production of recombinant hSGLT1 transporter in E. coli and P. pastoris was evaluated. A number of different expression vector c onstructs of hSGLT1 for its production in E. coli were prepared. Various affinity tags were appended to the transporter C-terminal to enable transporter detection and purification. We attempted expression of transporter in different expression vectors, in different conditions and in different E. coli strains but we could not get the expression of hSGLT1. During this pro cess, we constructed clone pET22b-Unc-F- hSGLT1 containing Unc-F gene (encoding the β -subunit of E. coli ATP synthase), in this case also we could not find out any hSGLT1 expression but also no expression of β -subunit of the E. coli ATP synthase, which was expressed in la rge amounts in the control clone (pET22b- Unc-F). This finding along with the fact th at hSGLT1 mRNA contains 5 RNase E sites suggests that in E. coli hSGLT1 mRNA is unstable and a ll mRNA is degraded by RNase E and other exoribonucleases before its translation into protein. Heterologous expression of hSGLT1 was also carried out in P. pastoris. hSGLT1 gene containing FLAG tag and a 10 amino acids long spacer at its C-terminal was cloned into pPICZB plasmid and the resulted expression vector pPICZB-hSGLT1 was transformed into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by Nickel- affinity-chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured P. pastoris cells. The purity of the hSGLT1 wa s more than 95% pure, as judged from Coomassie stained gels. Purified hSGLT1 migrates on SDS-PAGE at an apparent mass of 55 kDa, which corresponds to that of the nonglycosylated transporter. Reconstitution of purified hSGLT1 yields proteoliposomes active in Na + -dependent glucose uptake and sodium- stimulated uptake of 50 µM α-MDG is completely blocked by 10 µM phlorizin. The 1 min time course of α-MDG uptake in the proteoliposomes demonstrated a characteristic overshoot in the presence of Na+ , indicating that sugar was concentrated within the proteoliposomes and that uptake was energised by Na+ electrochemical gradient. In order to further characterize recombinant hSGLT1 in proteoliposomes, we performed competitive uptake studies of 100 µM α-MDG in the presence of inhibitor (100 µM phlorizin) high-affinity substrates (10 mM D-glucose and D-galactose) and low-affin ity substrates (10 mM 2-Deoxy-D-glucose, mannose, allose, and L- glucose) that inhib ited sugar uptake according to their affinity towards the transporter in cells and membranes. hSGLT1 in proteoliposomes exhibits substrate specificity in the following order, Phlz >> α-MDG ≈ D-Glc ≈ D-Gal >> 2Doglc > Man > All > L-Glc. Recombinant reconstituted hSG LT1 shows sterospecificity as its preferred D-Glu over L-Glu, D-Glu inhibited 75% of α-MDG uptake L-Glu, inhibited only Summary 90 2%. The equatorial orientation of the OH-group at the positions C-2 and C-3 of the D-glucose seems to be important for its recognition by hSGLT1 since the presence of mannose, 2- Deoxy-D-glucose and allose signi ficant inhibitory effect on α-MDG uptake was observed. The 78% inhibition of α-MDG uptake in the presence of D-galactose indicates the orientation of OH-group at C-4 (equatorial in D-glucose and axial in D-galactose) is not that important for their recognition by cotransporter because D-Glu and D-Gal inhibited sugar uptake by transporter upto equal extent. Phlz inhibited 84% of α-MDG uptake in proteoliposomes. Catalytic turnover of hSGLT1 based on the Vmax of 3.4 µmol × mg hSGLT1 -1 × min-1 is 6 s -1 . These results clearly indicate that purified recombinant hSGLT1 posses the full functionalities in this reconstituted system. For the identification of sugar-binding sites and phl orizin interactions sites in the transporter four new photoprobes were synthesized and s ubsequently evaluated for their effects on SGLT1 mediated α-MDG transport in rabbit small intestine brush border membrane vesicles. Results of these experiments shows that [(2’-Iodo -4’- ( 3 ’ ’ -trifluoromethyldiazirinyl) phenoxy]- D-glucopyranoside (TIPDG), [(4´-Benzoyl ) phenoxy]-D-glucopyranoside (BzG), 3- Azidophlorizin (3-AP), n-Methyl-6C-(Azimethyl)- D-glucopyranoside (6-AG) inhibit α-MDG uptake. The Ki values of TIPDG (22 ± 5µM) and its prototype compound arbutin (25 ± 6µM), BzG (12 ± 2µM) and its respective model compound phlorizin (8 ± 1.2µM), 3-AP (8.6 ± 1.6µM) and its parent compound phlorizin (8 ± 1.2µM) and of 6-AG (40 ± 5µM) and its sibling D-glucose (1 mM) were essentially id entical. Photoaffinity labeling were performed with a functional domain of SGLT1 (ami no acids 564-638 of the SGLT1 amino acid sequence, loop 13) with TIPDG, BzG, and 3- AP clearly demonstrated that our probes successfully labeled part of the SGLT1 protein. 3-AP was further used to identify phlorizin -binding sites in the truncated loop 13 by the combination of photoaffinity labeling, SDS- PAGE, enzymatic fragmentation, and MALDI- mass spectrometry analysis. The attachment site of the ortho position of ring B of phlorizin could be allocated to amino acid Arg-602, thus, an interaction of this region with the B ring of phlorizin can be assumed. Thereby the binding pocket could accommodate the phlorizin molecule in the previously predicted conformation. To summarize, this study provides first exampl e of large-scale functional expression and purification of full-length human Na + /D-glucose cotransporter. Recombinant hSGLT1 retains full functionalities when reconstituted into proteoliposomes. The ability to purify functional cotransporter enables a battery of biochemical and biophysical experiments to probe the structure and function of hSGLT1, with results po ssibly relevant to other cotransporters in the SGLT family. The synthesis of four new photoaffinity probes provides tools for the identification of sugar translocation pathway and inhibitor binding in the recombinant hSGLT1. Zusammenfassung 91 7. Zusammenfassung Der humane Na+ /D-Glucose Kotransporter (hSGLT1) gehört zur Transporerfamilie SCL (solute carrier) 5 und spielt eine zentrale Rolle bei der Homöostase von Glucose, Salz und Wasser. Ziel der vorliegenden Arbeit war zunächst die heterologe Expression des Transporters in E. coli und P. pastoris. Für die Expression in E. coli wurden verschiedene Expressionsvektoren verwendet und zur Detektion und Reinigung des Tran sporters unterschiedliche Affinitäts Tags an dessen C-Terminus angehängt. Wir haben die Expression des Transporters mit verschiedenen Expressionsvektoren und E. coli Stämmen unter zahlreichen Bedingungen versucht, ohne dass die Expression von hSGLT1 gelang. Während dieser Versuche haben wir den Klon pET22b-Unc-F-hSGLT1 hergestellt, welcher das Unc-F Gen für die β -Untereinheit der E. coli ATP Synthase enthält. In diesem Fall konnten weder hSGLT1 noch ATPase Expression nachgewiesen werden, obwohl letztere in hohem Ausmaß im Kontrollklon (pET22b-Unc-F) exprimiert wurde. Zusammen mit der Tatsache, dass die mRNA des hSGLT1 5 RNasE Sequenzen enthält, lässt dies den Schluß zu, dass die mRNA des hSGLT1 in E. coli instababil ist und dass die mRNA durch RNase E und andere Exoribonucleasen abgebaut wird, noch bevor die Translation zum Protein erfolgen kann. Desweiteren wurde die heterologe Expression von hSGLT1 in P. pastoris durchgeführt. Das hSGLT1 Gen wurde mit einem C-terminalen FLAG Tag und einem 10 Aminosäuren langen Spacer in das Plasmid pPICZB kloniert. M it dem erhaltenen Expressionsvektor pPICB- hSGLT1 wurde der P. pastoris Stamm GS115 durch Elektroporation transformiert. Durch Aufreinigung des hSGLT1 mittels Nickel-A ffinitätschromatographie konnten 3 mg gereinigtes rekombinantes hSGLT1 Protein pro Liter P. pastoris Kultur angereichert werden. Die Reinheit des hSGLT1 lag bei über 95%. Gereinigter hSGLT1 hat in SDS-PAGE Gelen ein scheinbares Molekulargewicht von 55 kDa, welches der Größe eines nicht-glykosilierten Transporters entspricht. Nach Rekonstitution des gereinigten SGLT1 in Proteoliposomen konnte eine natriumgetriebene Akkumulation von α-Methyl-D-Glucose ( α-MDG) nachgewiesen werden, die vollkommen durch 10 µM Phlorizin gehemmt wurde. Um rekombinanten hSGLT1 in Proteoliposomen näher zu charakterisieren, wurden kompetitive Aufnahmestudien mit 100 µM α-MDG in der Gegenwart von Inhibitor (100 µM Phlorizin), Substraten mit hoher Affinitä t (10 mM D-Glucose und D-Galactose) und Substraten mit geringer Affinität (10 mM 2-Deoxy-D-Glucose, Mannose, Allose und L- Glucose) durchgeführt, welche die Zuckeraufn ahme entsprechend ihrer Affinität zum nativen Transporter inhibierten. Zusammenfassung 92 hSGLT1 in Proteoliposomen weist folgende Substratspezifität auf: Phlz >> α-MDG ≈ D-Glc ≈ D-Gal >> 2DoGlc > Man > All > L-Glc. Die katalytische Umsatzrate des hSGLT1 beträgt 6 s -1 auf der Basis von Vmax = 3,4 µmol × mg hSGLT1 -1 × min-1 . Diese Ergebnisse zeigen deutlich dass gereinigter rekombinanter hSGLT1 in dem verwendeten System volle Funktionalität aufweist. Für die Identifizierung der Bindungsstellen des Transporters für Zucker und der Interaktionsstellen mit Phlorizin wurden vi er neue Photo-Proben synthetisiert und deren Wirkung auf den SGLT1-vermittelten α-MDG Transport in Vesikeln der Bürstensaummembran des Kaninchen-Dünndarms untersucht. Die Ergebnisse zeigten dass [(2’-Iodo -4’- ( 3 ’ ’ -trifluoromethyldiazirinyl) phenoxy]-D -glucopyranoside (TIPDG), [(4´- Benzoyl) phenoxy]-D- glucopyranoside (BzG), 3-Azidophlorizin (3-AP), n-Methyl-6C- (Azimethyl)-D- glucopyranoside (6-AG) die α-MDG Aufnahme inhibierten. Die Ki Werte der Ausgangsverbindungen und deren Derivate waren nahezu identisch; 25 ± 6 µM für Arbutin und 22 ± 5 µM für TIPDG, 8 ± 1,2 µM für die Ausgangsverbindung Phlorizin und 12 ± 2 µM für BzG und 8,6 ± 1,2 µM für 3-AP als deren Derivate und 1 mM für die Ausgangsverbindung D-Glucose und 40 ± 5 µM für 6-AG. Photoaffinitäts-Markierungen wurden mit eine r funktionellen, rekombinant exprimierten Domäne des SGLT1 (Aminosären 564-638 de s loop 13) mit TIPDG, BzG und 3-AP durchgeführt. Es konnte deutlich gezeigt werd en, dass die Proben erfolgreich Teile des SGLT1 Proteins markierten. Um die Phlorizin-Bindestellen des Loop 13 zu id entifizieren wurde im weiteren 3-AP in einer Kombination von Photoaffinitäts Markierungen, SDS-PAGE, enzymatischer Fragmentierung und MALDI Massenspektrometrie verwendet. Die Anlagerungsstelle des Ring B von Phlorizin konnte der Aminosäure Arg-602 zugeordnet werden. Folglich kann eine Wechselwirkung dieser Region mit dem Phlorizin B-Ring angenommen werden. Dadurch könnte die Bindungstasche das Phlorizinmolekül in der zuvor vorhergesagten Konformation beherbergen. In dieser Studie konnte zum ersten mal der humane Na+ /D-Glucose Kotransporter in großem Maßstab funktionell exprimiert und gereinigt werden. 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Dortmund, den 25.02.05 Navneet Kumar Tyagi Acknowledgements Acknowledgements I take this opportunity to convey my deepest sense of gratitude to all those who helped in my venture. First and foremost, I would like to thank Prof. Dr. Rolf K.H. Kinne for his ardent support and guidance throughout my PhD career. It is indeed an honor to have carried out my doctoral research under his supervision. He has introduced me to a multitude of fields including the most exciting field of membrane protein biochemistry and molecular biology. The experience has been an enlightening one, thereby making me a penchant for various aspects of this field, besides being cognizant of other areas of studies carried out at the interface of chemistry and biology. He has always lended me a patient and helping attitude. Many a time it has been his elegant creativity and grand experience in the field of science that helped me tackle the problem with ease. Thanks to his timely suggestions at various stages of this work, which has truly helped and managed to mould the work undertaken into its present shape. I am also grateful to late Prof. Dr. Anil K. Lala (Indian Institute of Technology, Bombay, India) for inculcating in me, a working philo sophy that is necessary for doing cutting edge research. I have gained an invaluable experience through my association with him, on how to approach, how not to and finally tackle the problem at hand. My special thanks to Prof. Dr. Wolfgang Siess (Ludwing Maxmillian University, Munich, Germany) for his kind permission to perform my experiments related to the expression of human sodium/glucose cotransporter in his lab. His immense support and encouragements has helped me to succeed in high-level expression of this protein. I thank Prof. Dr. Keith W. Miller (Harvard Medical School/ Massachusetts General Hospital, Boston, USA), and all his group member fo r a wonderful collaboration and supportive discussions. I thank Prof. Dr. Herbert Waldmann for being in my examination committee. I wish to thank Dr. Pankaj Goyal for his wonde rful advices and support for the expression of hSGLT1 protein, without whom this work would not have taken a complete shape. Special thanks to my expert molecular biologist friends Dr.Patrick R. D`Silva, Dr. Rana Roy, and Dr. Suneel Kateriya for their valuable advices. I wish to thank Dr. Jutta Rötter for her support, and managing all the bureaucratic matters especially money matters for me. I thank International Max-Planck Research School in Chemical Biology for providing me fellowship and for generous travel grants. I thank Natascha and Niklas for their assistance in official matters and for giving me loads of information. I wish to thank my special friends Manish Ve rma and Deepali Gupta for their encouragement, advices, affection and support. I have spent three wonderful and memorable years with them. I wish to thank Dr. Ritu Dhawan for carefully proofreading the entire thesis. Acknowledgements I wish to thank all my friends namely, Dharemendra Pandey, Dr. Heidrun Olsen, Dr. Okram Barun, Dr. Anupam Bhattacharya, Dr. Ankur Kuls herstha and all others for their excellent support. I thank all the staff members of the Epithelial cell physiology department for their kind assistance during the course of this work. I owe to my parents for their continuous support and blessings. Special appreciations are due to my loving brothers, Vikas, Adarsh and A zad, bhabhis, Vineta and Bina, my wonderful sister, Poonam and Jijaji subodh, for their love and care bestowed upon me. I cannot forget affection given by Abhinav (Vishu), Appoorva (Chinu), Soumya (Anya), Abhijay (Abhi), Saahil (Arnav) and Aryan. Curriculum vitae Curriculum Vitae Name: Navneet Kumar Tyagi Date of birth: July 13, 1977 Place of birth Meerut, India Nationality Indian Education: July 1990- June 1992 10 th Std. (St. Charls Inter College Sardhana, India) July 1992- June 1994 12 th Std. (St. Charls Inter College Sardhana, India) July 1994- June 1997 BS, Biology (SGPG College, Saraurpur Khurd, India) July 1997-June 1999 MS, Chemistry (University of Roorkee, Roorkee, India) July 1999- June 2001 Project Assistant in the Department of Chemistry, Indian Institute of Technology, Bombay, India Nov. 2001- present Ph. D Thesis at the Department of Epithelial Cell Physiology, Max-Planck Institute for Molecular Physiology, Dortmund, Germany, Under the Supervision of Prof. Dr. Dr. h.c. Rolf K.H. Kinne A wards/Achievements and experiences: 2004 Worked for 9 months Collaboration project with Prof. Dr. Wolfgang Siess , Institute for Prevention of Cardiovascular Diseases in Ludwig Maximilians University Munich, Germany 2003 Worked for 5 months Collaboration project with Prof. Keith W. Miller . in the department of Anesthesia and Critical care, Massachusetts General Hospital/Harvard Medical School, Boston, USA. 2003 Recipient of Martin-Schmeisser fe llowship from university of Dortmund Germany 2002-2004 Awarded fellowship for Ph. D program by International Max-Planck Research School in Chemical Biology. 2001 Worked as a Visiting scientist in De partment of Neurobiology, Heidelberg University, Germany. 1999-2001 Worked as a junior research assist ant in Indian Institute of Technolgy, Bombay, India. 1999 Awarded Junior Research Fellowship by Department of Science and Technology, Government of India. Curriculum vitae 1999 Qualified for Graduate aptitude test in Engineering (GATE) with score of 94.43 in the year 1999. Publications: 1. Navneet K. Tyagi ; and Rolf K. H. Kinne (2003) “Synt hesis of Photoaffinity Probe [(2'- Iodo-4'-(3''-trifluormethyldiazirinyl) Phenoxy]- D-Glucopyranoside (TIPDG) and [(4'- Benzoyl) Phenoxy)]- D-Glucopyranoside for the Identification of Sugar Binding and Phlorizin Binding Sites in the Sodium/ D-Glucose Cotransporter Protein (SGLT1).” Anal. Biochem. 3 2 3, 74-83. 2. M. Mobeen. Raja; Navneet K. Tyagi ; and Rolf K. H. Kinne (2003) “Phlorizin Recognition in a C-terminal Fragments of SGLT1 Studied by Trp Scanning and Affinity Labeling.” J. Biol. Chem. 278, 49154-49163. 3. Navneet K. Tyagi ; Pankaj Goyal; Dharemendra Pandey; Wolfgang Siess; and Rolf K. H. Kinne (2005) “Functional Expression, Puri fication, and Characterization of Human Na+ / D-Glucose Transporter (hSGLT1) in Pichia pastoris ” J. Biol. Chem. (Communicated). 4. Navneet K. Tyagi ; Pankaj Goyal; Wolfgang Siess; and Rolf K. H. Kinne (2005) “Messenger RNA Stability is the Major Hurdle in the Expression of Human Membrane Proteins in E. coli ” (Under Preparation). 5. A zad Kumar; Navneet K. Tyagi ; Pankaj Goyal; Wolfgang Siess; and Rolf K. H. Kinne (2005) “Ligand-Dependent C onformational Changes in Human Sodium/Glucose Cotransporter1 in Soluti on and in Reconstituted Forms as Studied by Fluorescence Spectroscopy” (Under Preparation). 6. Dharemendra Pandey; Pankaj Goyal; Navneet K. Tyagi ; Rolf K. H. Kinne; and Wolfgang Siess (2005) “Over-expressi on, Purification, and Functional Characterization of Human LIMK-2 in the Yeast, Pichia pastoris ” (Under Preparation). 7. Pankaj Goyal; Dharemendra Pandey; Navneet K. Tyagi ; Rolf K. H. Kinne; and Wolfgang Siess (2005) “Identification and Ch aracterization of Nuclear and Nucleolus Signal Sequences in LIMK2” (Under Preparation).