Ethnicity-dependent Polymorphism in Na+-taurocholate Cotransporting Polypeptide (SLC10A1) Reveals a Domain Critical for Bile Acid Substrate Recognition
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m305782200
ISSN1083-351X
AutoresRichard Ho, Brenda F. Leake, Richard Roberts, Wooin Lee, Richard B. Kim,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoThe key transporter responsible for hepatic uptake of bile acids from portal circulation is Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1). This transporter is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function. Therefore, functionally relevant polymorphisms in this transporter would be predicted to have an important impact on bile acid homeostasis/liver function. However, little is known regarding genetic heterogeneity in NTCP. In this study, we demonstrate the presence of multiple single nucleotide polymorphisms in NTCP in populations of European, African, Chinese, and Hispanic Americans. Specifically four nonsynonymous single nucleotide polymorphisms associated with a significant loss of transport function were identified. Cell surface biotinylation experiments indicated that the altered transport activity of T668C (Ile223 → Thr), a variant seen only in African Americans, was due at least in part to decreased plasma membrane expression. Similar expression patterns were observed when the variant alleles were expressed in HepG2 cells, and plasma membrane expression was assessed using immunofluorescence confocal microscopy. Interestingly the C800T (Ser267 → Phe) variant, seen only in Chinese Americans, exhibited a near complete loss of function for bile acid uptake yet fully normal transport function for the non-bile acid substrate estrone sulfate, suggesting this position may be part of a region in the transporter critical and specific for bile acid substrate recognition. Accordingly, our study indicates functionally important polymorphisms in NTCP exist and that the likelihood of being carriers of such polymorphisms is dependent on ethnicity. The key transporter responsible for hepatic uptake of bile acids from portal circulation is Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1). This transporter is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function. Therefore, functionally relevant polymorphisms in this transporter would be predicted to have an important impact on bile acid homeostasis/liver function. However, little is known regarding genetic heterogeneity in NTCP. In this study, we demonstrate the presence of multiple single nucleotide polymorphisms in NTCP in populations of European, African, Chinese, and Hispanic Americans. Specifically four nonsynonymous single nucleotide polymorphisms associated with a significant loss of transport function were identified. Cell surface biotinylation experiments indicated that the altered transport activity of T668C (Ile223 → Thr), a variant seen only in African Americans, was due at least in part to decreased plasma membrane expression. Similar expression patterns were observed when the variant alleles were expressed in HepG2 cells, and plasma membrane expression was assessed using immunofluorescence confocal microscopy. Interestingly the C800T (Ser267 → Phe) variant, seen only in Chinese Americans, exhibited a near complete loss of function for bile acid uptake yet fully normal transport function for the non-bile acid substrate estrone sulfate, suggesting this position may be part of a region in the transporter critical and specific for bile acid substrate recognition. Accordingly, our study indicates functionally important polymorphisms in NTCP exist and that the likelihood of being carriers of such polymorphisms is dependent on ethnicity. Bile acids, synthesized from the enzymatic catabolism of cholesterol, are the major solutes in bile, essential for the maintenance of bile flow and biliary lipid secretion (1St. Pierre M.V. Kullak-Ublick G.A. Hagenbuch B. Meier P.J. J. Exp. Biol. 2001; 204: 1673-1686Crossref PubMed Google Scholar). In addition, an important mechanism for cholesterol homeostasis occurs through its elimination in the form of bile acids. Indeed de novo synthesis of bile acids from cholesterol is thought to account for nearly half of the daily elimination of cholesterol from the body (1St. Pierre M.V. Kullak-Ublick G.A. Hagenbuch B. Meier P.J. J. Exp. Biol. 2001; 204: 1673-1686Crossref PubMed Google Scholar). In the gastrointestinal tract, bile acids also modulate the release of pancreatic secretions and gastrointestinal peptides and activate enzymes required for the absorption of lipid-soluble vitamins (2Koop I. Schindler M. Bosshammer A. Scheibner J. Stange E. Koop H. Gut. 1996; 39: 661-667Crossref PubMed Scopus (44) Google Scholar, 3Riepl R.L. Fiedler F. Ernstberger M. Teufel J. Lehnert P. Eur. J. Med. Res. 1996; 1: 499-505PubMed Google Scholar). Furthermore, their detergent properties assist solubilization of cholesterol and dietary fats in the intestine. Bile salts are efficiently reabsorbed in the small intestine and are returned to the liver via the portal circulation and resecreted into bile, thus forming an enterohepatic circuit (4Trauner M. Boyer J.L. Physiol. Rev. 2003; 83: 633-671Crossref PubMed Scopus (798) Google Scholar). The efficient enterohepatic recirculation of bile acids is maintained by polarized expression of bile acid uptake and efflux transporters in the intestine and liver (4Trauner M. Boyer J.L. Physiol. Rev. 2003; 83: 633-671Crossref PubMed Scopus (798) Google Scholar). Moreover, taurine or glycine conjugates of bile acids tend to be polar and hydrophilic, thus dependent on transporter proteins for cellular uptake and efflux (5Hofmann A.F. Feldman M. Scharschmidt B.F. Sleisenger M.H. Gastrointestinal and Liver Disease. Saunders, Philadelphia1998: 937-948Google Scholar). In the liver, it is estimated that Na+-dependent transport pathways account for greater than 80% of the hepatic uptake of conjugated bile acids such as taurocholate (6Muller M. Jansen P.L. J. Hepatol. 1998; 28: 344-354Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 7Anwer M.S. Hegner D. Hoppe-Seyler's Z. Physiol. Chem. 1978; 359: 181-192PubMed Google Scholar, 8Scharschmidt B.F. Stephens J.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 986-990Crossref PubMed Scopus (89) Google Scholar, 9Yamazaki M. Suzuki H. Hanano M. Sugiyama Y. Am. J. Physiol. 1993; 264: G693-G701PubMed Google Scholar, 10Kouzuki H. Suzuki H. Ito K. Ohashi R. Sugiyama Y. J. Pharmacol. Exp. Ther. 1998; 286: 1043-1050PubMed Google Scholar). The transporter responsible for the observed Na+-dependent uptake of conjugated bile salts is Na+-taurocholate cotransporting polypeptide (NTCP, 1The abbreviations used are: NTCP, Na+-taurocholate cotransporting polypeptide; SNP, single nucleotide polymorphism; ISBT, ileal Na+-dependent bile acid transporter; BSEP, bile salt export pump; PBS, phosphate-buffered saline. 1The abbreviations used are: NTCP, Na+-taurocholate cotransporting polypeptide; SNP, single nucleotide polymorphism; ISBT, ileal Na+-dependent bile acid transporter; BSEP, bile salt export pump; PBS, phosphate-buffered saline. SLC10A1) (11Hagenbuch B. Stieger B. Foguet M. Lubbert H. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10629-10633Crossref PubMed Scopus (451) Google Scholar, 12Cattori V. Eckhardt U. Hagenbuch B. Biochim. Biophys. Acta. 1999; 1445: 154-159Crossref PubMed Scopus (49) Google Scholar, 13Kramer W. Stengelin S. Baringhaus K.H. Enhsen A. Heuer H. Becker W. Corsiero D. Girbig F. Noll R. Weyland C. J. Lipid Res. 1999; 40: 1604-1617Abstract Full Text Full Text PDF PubMed Google Scholar, 14Hagenbuch B. Meier P.J. J. Clin. Investig. 1994; 93: 1326-1331Crossref PubMed Scopus (396) Google Scholar). This bile acid uptake transporter, whose function is coupled to a sodium gradient (15Weinman S.A. Yale J. Biol. Med. 1997; 70: 331-340PubMed Google Scholar), is expressed exclusively in the liver and localized to the basolateral membrane of the hepatocyte (16Stieger B. Hagenbuch B. Landmann L. Hochli M. Schroeder A. Meier P.J. Gastroenterology. 1994; 107: 1781-1787Abstract Full Text PDF PubMed Google Scholar). The human NTCP gene encodes a 349-amino acid protein (14Hagenbuch B. Meier P.J. J. Clin. Investig. 1994; 93: 1326-1331Crossref PubMed Scopus (396) Google Scholar) and shares 77% amino acid sequence identity with rat Ntcp (17Meier P.J. Eckhardt U. Schroeder A. Hagenbuch B. Stieger B. Hepatology. 1997; 26: 1667-1677Crossref PubMed Scopus (323) Google Scholar). Hagenbuch et al. (18Hagenbuch B. Scharschmidt B.F. Meier P.J. Biochem. J. 1996; 316: 901-904Crossref PubMed Scopus (110) Google Scholar) demonstrated that, when Xenopus laevis oocytes were coinjected with total rat liver mRNA and antisense oligonucleotides specific to Ntcp, the expressed Na+-dependent taurocholate transport activity was reduced by 95%. This finding suggests a potentially central role for Ntcp in the hepatic uptake of bile acids. Accordingly, the extent of its expression or function would be predicted to significantly affect enterohepatic circulation of bile acids and directly affect cellular signaling pathways importantly involved in cholesterol homeostasis and hepatocyte function. One potential source of altered NTCP function may be genetic heterogeneity in this transporter related to the presence of single nucleotide polymorphisms, or SNPs. Indeed, functional polymorphisms are known to exist among bile acid transporter family members. For example, mutations in the ileal Na+-dependent bile acid transporter (ISBT, SLC10A2) have been identified (19Oelkers P. Kirby L.C. Heubi J.E. Dawson P.A. J. Clin. Investig. 1997; 99: 1880-1887Crossref PubMed Scopus (305) Google Scholar). This transporter, expressed on the apical surface of ileal enterocytes and mediating the Na+-dependent uptake of conjugated and unconjugated bile acids in the intestine (20Craddock A.L. Love M.W. Daniel R.W. Kirby L.C. Walters H.C. Wong M.H. Dawson P.A. Am. J. Physiol. 1998; 274: G157-G169PubMed Google Scholar, 21Shneider B.L. Dawson P.A. Christie D.M. Hardikar W. Wong M.H. Suchy F.J. J. Clin. Investig. 1995; 95: 745-754Crossref PubMed Google Scholar, 22Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar), shares significant sequence homology with NTCP (23Dawson P.A. Oelkers P. Curr. Opin. Lipidol. 1995; 6: 109-114Crossref PubMed Scopus (68) Google Scholar, 24Hallen S. Branden M. Dawson P.A. Sachs G. Biochemistry. 1999; 38: 11379-11388Crossref PubMed Scopus (43) Google Scholar). Interestingly, certain loss of function mutations in the coding regions of ISBT result in a syndrome of primary bile acid malabsorption (19Oelkers P. Kirby L.C. Heubi J.E. Dawson P.A. J. Clin. Investig. 1997; 99: 1880-1887Crossref PubMed Scopus (305) Google Scholar), characterized by severe diarrhea, malabsorption of fat, and malnutrition. Furthermore, a number of mutations in the bile salt export pump (BSEP, ABCB11), the ATP-dependent bile salt efflux transporter localized to the canalicular membrane of hepatocytes (25Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. J. Biol. Chem. 1998; 273: 10046-10050Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar), have been linked to progressive familial intrahepatic cholestasis type 2 (26Jansen P.L. Strautnieks S.S. Jacquemin E. Hadchouel M. Sokal E.M. Hooiveld G.J. Koning J.H. Jager-Krikken A. Kuipers F. Stellaard F. Bijleveld C.M. Gouw A. Van Goor H. Thompson R.J. Muller M. Gastroenterology. 1999; 117: 1370-1379Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 27Strautnieks S.S. Bull L.N. Knisely A.S. Kocoshis S.A. Dahl N. Arnell H. Sokal E. Dahan K. Childs S. Ling V. Tanner M.S. Kagalwalla A.F. Nemeth A. Pawlowska J. Baker A. Mieli-Vergani G. Freimer N.B. Gardiner R.M. Thompson R.J. Nat. Genet. 1998; 20: 233-238Crossref PubMed Scopus (848) Google Scholar), a progressive cholestatic liver disease characterized by loss of biliary bile acid secretion and an absence of BSEP expression on the canalicular membrane. However, little is known regarding genetic heterogeneity in NTCP. The only published data to date have been a report of two SNPs in the coding region of NTCP in the Japanese population, conducted as a part of a large scale SNP discovery effort but lacking any functional studies of identified SNPs (28Saito S. Iida A. Sekine A. Ogawa C. Kawauchi S. Higuchi S. Nakamura Y. J. Hum. Genet. 2002; 47: 576-584Crossref PubMed Scopus (52) Google Scholar). Clearly, loss of function mutations in NTCP, if found to be present, would significantly add to our knowledge of the genetic basis of altered bile acid absorption, cholesterol elimination, and hepatocyte function. In this report we present data that support the presence of ethnicity-dependent functionally deleterious polymorphisms in NTCP. Moreover, functional studies indicate the presence of a discrete region within NTCP that is essential for bile acid substrate recognition. Materials—[3H]Taurocholate (3.4 Ci/mmol, >97% purity), unlabeled taurocholate, [3H]cholate (55 Ci/mmol, >97% purity), unlabeled cholate, [3H]estrone sulfate (53 Ci/mmol, >97% purity), and unlabeled estrone sulfate were purchased from PerkinElmer Life Sciences. Recombinant vaccinia virus containing the T7 RNA polymerase gene (vtf-7) was a gift provided by Dr. Bernard Moss (National Institutes of Health, Bethesda, MD). The pEF6/V5-His-TOPO® expression vector was purchased from Invitrogen. The monoclonal mouse anti-V5 antibody was purchased from Invitrogen. Human liver samples were kindly provided by Dr. F. P. Guengerich at our institution. Genomic DNA isolated from peripheral blood lymphocytes of healthy European American, African American, Chinese American, and Hispanic American volunteers was purchased from Coriell Cell Repositories (Camden, NJ). All other chemical and reagents, unless stated otherwise, were obtained from Sigma and were of the highest grade available. Identification of SNPs in NTCP—Initially, total genomic DNA was isolated from human liver samples (Nashville Regional Organ Procurement Agency, Nashville, TN) from 20 European American donors without any history of underlying liver pathology or prior medications via the DNeasy® tissue kit (Qiagen, Valencia, CA) and quantified using an MBA 2000 mass spectrophotometer (PerkinElmer Life Sciences). Genomic DNA isolated from peripheral blood lymphocytes of 30 additional European American, 50 African American, and 50 Chinese American healthy volunteers was obtained from Coriell Cell Repositories. A 226-kilobase bacterial artificial chromosome clone (CNS01RG6) containing the complete intron-exon boundaries for the human NTCP gene was identified from the Human Genome Project (GenBank™). Primer pairs to amplify each of the five exonic regions of NTCP were designed and are as follows: exon 1, forward 5′-GAACTGCACAAGAAACGGAGTC-3′ and reverse 5′-GCTCTTCCCCTCAATTGTGAC-3′; exon 2, forward 5′-GCACTTAGCAGGCACTCAAC-3′ and reverse 5′-GACAGTGAATCCTTAGAGTGC-3′; exon 3, forward 5′-CCAATTTCTACCTGTGCTTCC-3′ and reverse 5′-CTGCAGTGAGCTGAGAATGTG-3′; exon 4, forward 5′-CAGCACTGGGACAAAGTTGC-3′ and reverse 5′-GGCTCAGGTCTAATATTGGAG-3′; exon 5, forward 5′-CACACTCTAGGGCTGAGTTG-3′ and reverse 5′-GCTTCTTGGGTAGACACCCTGTC-3′. Using a GeneAmp® PCR system 9700 (Applied Biosystems, Foster City, CA), PCR was carried out using ∼200 ng of human liver genomic DNA for European American subjects or ∼25 ng of genomic DNA (Coriell Cell Repositories) previously digested with the restriction endonuclease XhoI (New England Biolabs, Inc., Beverly, MA) for additional European Americans and all African American and Chinese American subjects consisting of dNTPs (0.25 mm each), the specific primer pair (4 μm each), 50 mm KCl, 10 mm Tris-HCl, 2.5 mm MgCl2, and 2.5 units of AmpliTaq® DNA polymerase (PerkinElmer Life Sciences) in a final reaction volume of 50 μl. PCR was generally carried out at 94 °C for 30 s, 55 °C for 20 s, and 72 °C for 20 s for 30 cycles with the exception of exon 2, which was carried out at 94 °C for 30 s, 58 °C for 5 s, and 72 °C for 10 s for 30 cycles. PCR products of expected sizes were completely visualized using ethidium bromide-stained 2% agarose gels and were fully sequenced in all subjects with an ABI 3700 DNA analyzer (Applied Biosystems). SNPs were identified utilizing the computer software program AlignX (Vector NTI, Version 7.0, InforMax, Inc., Frederick, MD) and confirmed by direct visualization of the sequences. A search of available SNP data bases 2Data bases included IMS-JST Japanese SNP (snp.ims.u-tokyo.ac.jp/), dbSNP (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = snp), PharmGKB (www.pharmgkb.org), and GeneCards™ (bioinfo.weizmann.ac.il/cards/). was performed to identify any additional polymorphisms. Wild-type and Variant NTCP Plasmid Construction—The full open reading frame of human NTCP cDNA was obtained by PCR using AmpliTaq® DNA polymerase (PerkinElmer Life Sciences) from a cDNA library synthesized from human liver mRNA using oligonucleotide primers 5′-ATGGAGGCCCACAACGCGTCT-3′ as the forward and 5′-CTAGGCTGTGCAAGGGGA-3′ as the reverse. A single PCR product of expected size was visualized on an ethidium bromide-stained 1.2% agarose gel. An aliquot of the PCR product was ligated into the pEF6/V5-His-TOPO® vector (Invitrogen). After transformation and growth in Escherichia coli, individual colonies containing the pEF6/V5-His-TOPO®/NTCP construct were identified. A pEF6/V5-His-TOPO®/NTCP with the NTCP cDNA inserted in the sense orientation downstream from the T7 promoter region was fully sequenced using an ABI 3700 DNA analyzer (Applied Biosystems Inc.) and found to fully match the published reference sequence (GenBank™ accession number NM_003049) (14Hagenbuch B. Meier P.J. J. Clin. Investig. 1994; 93: 1326-1331Crossref PubMed Scopus (396) Google Scholar). This clone was termed NTCP*1. Site-directed mutagenesis was utilized to create the identified nonsynonymous allelic variants: C800T and T836C identified from Chinese American samples, A940G identified from a Hispanic American sample, and T668C identified from African American samples. The appropriate point mutations were introduced individually into wild-type NTCP (*1) packaged into pEF6/V5-His-TOPO® using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the following oligonucleotide primers: C800T, forward 5′-CAAAATGTCCAACTCTGTTTCACCATCCTCAATGTGGCCTTTC-3′ and reverse 5′-GAAAGGCCACATTGAGGATGGTGAAACAGAGTTGGACATTTTG-3′; T668C, forward 5′-GCCATGACACCACTCTTGACTGCCACCTCCTCCCTGATG-3′ and reverse 5′-CATCAGGGAGGAGGTGGCAGTCAAGAGTGGTGTCATGGC-3′; T836C, forward 5′-GGCCTTTCCACCTGAAGTCACTGGACCACTTTTCTTCTTTC-3′ and reverse 5′-GAAAGAAGAAAAGTGGTCCAGTGACTTCAGGTGGAAAGGCC-3′; A940G, forward 5′-GAGAAATTCAAGACTCCCGAGGATAAAACAAAAATG-3′ and reverse 5′-CATTTTTGTTTTATCCTCGGGAGTCTTGAATTTCTC-3′. The presence of the mutations was verified by full sequencing. Wild-type and variant NTCP were renamed the following for expression studies: wild-type, NTCP*1; C800T, NTCP*2; T668C, NTCP*3; T836C, NTCP*4; and A940G, NTCP*5. Determination of Genotypic Frequencies—Direct sequencing of PCR products was performed to determine the genotypic frequencies of the nonsynonymous polymorphisms C800T, T668C, T836C, and A940G. PCR was carried out as previously described utilizing genomic DNA digested previously with the restriction endonuclease XhoI (New England Biolabs, Inc.) from a total of 90 European American, 90 African American, 90 Hispanic American, and 100 Chinese American healthy volunteers (Coriell Cell Repositories) with primers to amplify exon 3 and exon 4 of the NTCP gene. Single PCR products of expected sizes were visualized on ethidium bromide-stained 2% agarose gels. PCR products were fully sequenced in all subjects using an ABI 3700 DNA analyzer (Applied Biosystems Inc.). Allele frequencies were calculated based upon the Hardy-Weinberg equilibrium. Cell Culture and Virus Preparation—HeLa (American Type Culture Collection) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin. For preparation of a viral stock of vtf-7 virus, HeLa cells grown to near confluency in 25-cm tissue culture plates were infected with 1 plaque-forming unit/10 cells. After an incubation period of 48 h at 37 °C, the infected cells were pelleted, homogenized, and recovered through centrifugation followed by titering of viral stock as described by Blakely et al. (29Blakely R.D. Clark J.A. Rudnick G. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar). Human hepatoma (American Type Culture Collection) cells (HepG2) were cultured in Dulbecco's modified Eagle's medium supplemented with l-glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin. For transient transfection, cells were grown on sterile uncoated 35-mm glass-bottomed microwell dishes (MatTek, Ashland, MA) and transfected at 70-80% confluency with 4 μg of V5-tagged NTCP plasmid DNA (NTCP*1, -*2, or -*3) using LipofectAMINE 2000 (Invitrogen). After 48 h, cells were analyzed by immunofluorescence confocal microscopy. Transport Studies Using Recombinant Vaccinia Virus—HeLa cells grown in 12-well plates (∼0.8 × 106 cells/well) were infected with vaccinia (vtf-7) at a multiplicity of infection of 10 plaque-forming units/cell in serum-free Opti-MEM I medium (Invitrogen) and allowed to adsorb for 30 min at 37 °C. Cells in each well were then transfected with 1 μg of wild-type or variant NTCP cDNA packaged into pEF6/V5-His-TOPO® vector (Invitrogen) along with Lipofectin® (Invitrogen) and incubated at 37 °C for 16 h. The parental plasmid lacking any insert was used as control. Transport was then evaluated using labeled substrates as outlined previously (30Kim R.B. Leake B. Cvetkovic M. Roden M.M. Nadeau J. Walubo A. Wilkinson G.R. J. Pharmacol. Exp. Ther. 1999; 291: 1204-1209PubMed Google Scholar). To measure the taurocholate, cholate, and estrone sulfate transport kinetics, radiolabeled substrate uptake during the linear phase (first 3 min) was assessed in the presence of various concentrations of unlabeled compound. Passive diffusion was determined by carrying out parallel experiments using the parental plasmid DNA lacking the transporter cDNA, and this value was then subtracted from the total uptake rate seen in the presence of the transporter cDNA. Michaelis-Menten-type nonlinear curve fitting was carried out to obtain estimates of the maximal uptake rate (Vmax) and the concentration at which half the maximal uptake occurs (Km) (Prism™, GraphPad, San Diego, CA). All experiments were carried out in duplicate on at least 2-3 experimental days. Epitope-tagged Wild-type and Variant NTCP Construction—Because of a lack of a high affinity antibody to human NTCP, we utilized a 14-amino acid epitope (V5) in the pEF6/V5-His-TOPO® vector (Invitrogen) to generate V5-tagged wild-type and variant NCTP proteins. The QuikChange® site-directed mutagenesis kit (Stratagene) was used to introduce a point mutation that converted the stop codon of NTCP to a lysine residue (TAG → AAG) utilizing the following oligonucleotide sense and antisense primers: 5′-CTGCTCCCCTTGCACAGCCAAGAAGGGCAATTCTGCAG-3′ and 5′-CTGCAGAATTGCCCTTCTTGGCTGTGCAAGGGGAGCAG-3′. The presence of the mutation was verified by full sequencing. The V5 tag was generated for wild-type NTCP and each of the nonsynonymous NTCP variants, and these were used for characterization of total protein and cell surface expression. NTCP Expression in HeLa Cells—HeLa cells transfected with NTCP cDNA tagged with the V5 epitope were scraped off plates, and the resulting suspension was centrifuged at 21,000 × g for 3 min. The cell pellet was reconstituted with HED buffer (25 mm HEPES, 1.5 mm EDTA, 1 mm dithiothreitol, pH 7.4) containing protease inhibitors (Complete, Roche Applied Science) and lysed by sonication. Samples were diluted with Laemmli buffer, and 3.75 μg of total cell protein were separated by SDS-PAGE on 10% gels. Following transfer onto nitrocellulose membranes, blots were probed with a monoclonal anti-V5 antibody (1:5000 dilution) (Invitrogen) and appropriate secondary antibody. To normalize sample loading, blots were stripped and reprobed with anti-calnexin antibody (StressGen, Victoria, British Columbia, Canada). Bands were visualized using enhanced chemiluminescence (Amersham Biosciences). NTCP Cell Surface Expression—HeLa cells were grown on 6-well plates and transfected with the NTCP cDNAs tagged with the V5 epitope using a similar protocol for transport experiments. Sixteen hours post-transfection, cells were washed with ice-cold phosphate-buffered saline Ca2+/Mg2+ (138 mm NaCl2, 2.7 mm KCl, 1.5 mm KH2PO4, 9.6 mm Na2HPO4, 1 mm MgCl2, 0.1 mm CaCl2, pH 7.3) and then treated with a membrane-impermeable biotinylating agent (sulfo-N-hydroxysuccinimide-SS-biotin, 1.5 mg/ml, Pierce) at 4 °C for 1 h. Subsequently the cells were washed three times with ice-cold phosphate-buffered saline Ca2+/Mg2+ containing 100 mm glycine and then incubated for 20 min at 4 °C with the same buffer to remove the remaining labeling agent. After washing with phosphate-buffered saline Ca2+/Mg2+, cells were disrupted with 700 μl of lysis buffer (10 mm Tris base, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Triton X-100, pH 7.4) containing protease inhibitors (Complete, Roche Applied Science) at 4 °C for 1 h with constant agitation. Following centrifugation, 140 μl of streptavidin-agarose beads (Pierce) was added to 600 μl of cell lysate and incubated for 1 h at room temperature. Beads were washed four times with ice-cold lysis buffer, and the biotinylated proteins were released by incubation of the beads with 2× Laemmli buffer for 30 min at room temperature. Similar to total cell lysates, samples of the biotinylated fractions (25 μl) were subjected to Western analysis for detection of immunodetectable NTCP with monoclonal anti-V5 antibody (1:5000 dilution) and the intracellular, endoplasmic reticulum-resident protein calnexin as described previously. Deglycosylation of Total and Cell Surface-expressed NTCP—HeLa cells were grown on 6-well plates and transfected with the wild-type NTCP cDNA tagged with the V5 epitope using a protocol similar to that for transport experiments. Total and cell-surface expressed fractions of wild-type NTCP (*1) were isolated as described previously and subjected to enzymatic deglycosylation (Glyko, San Leandro, CA). Briefly 15 μl of total or cell-surface expressed NTCP*1 was dissolved in 30 μl of deionized water. 10 μl of 5× incubation buffer (0.25 m sodium phosphate, pH 7.0) and 2.5 μl of denaturation solution (2% SDS and 1 m β-mercaptoethanol) were added to the proteins, gently mixed, and incubated at 100 °C for 5 min. After cooling to room temperature, 2.5 μl of detergent solution (15% Nonidet P-40) was added to the proteins. Then 1 μl each of N-glycanase, sialidase A, and O-glycanase or 3 μl of water (control) was added to the proteins and subsequently incubated for 3 h at 37 °C. Samples were diluted with 2× Laemmli buffer and subjected to Western analysis by SDS-PAGE on 12% gels for detection of immunodetectable NTCP with monoclonal anti-V5 antibody (1:5000 dilution). Immunofluorescence Confocal Microscopy—HepG2 cells transiently transfected with V5-tagged NTCP plasmid DNA were fixed for 10 min in ice-cold 70% methanol. After a 5-min wash in PBS, cells were permeabilized for 10 min in PBT (0.3% Triton X-100 in PBS, pH 7.4). After a 5-min wash in PBS, cells were placed in blocking buffer (2% bovine serum albumin in PBS) for 1 h at room temperature. Cells were then incubated in primary antibody (anti-V5 antibody diluted 1:500 in blocking buffer) for 2 h at room temperature. After three 5-min washes in PBST (0.05% Tween 20 in PBS, pH 7.4), cells were then incubated with secondary fluorescent dye (Texas Red)-labeled goat anti-mouse whole antibody (Molecular Probes, Eugene, OR) for 30 min at 37 °C. After three 5-min washes in PBST, cells were placed in PBS and viewed by confocal microscopy. HepG2 cells transfected with plasmid alone and cells transfected with V5-tagged NTCP wild-type plasmid DNA without incubation in primary antibody were used as two separate controls. Confocal microscopy was performed with a Zeiss Axiovert 100-M inverted microscope equipped with a LSM510 laser scanning unit. A Zeiss 63× 1.4 numerical aperture plan Apochromat oil immersion objective was used for all experiments. Confocal images were obtained using single excitation (595 nm) and emission (610-630 nm Texas Red) filter sets. For slow frame scanning, confocal images were obtained by scanning either laterally (top view, x-y scans) or axially (side view, x-z scans) across the cell. Image analysis and processing were performed with Zeiss LSM and Adobe Photoshop software. Statistical Analysis—Determination of the statistical differences between various group parameters was determined using either Student's t test, Mann-Whitney U test, analysis of variance (using Tukey-Kramer multiple comparison test), or Fisher's exact test as appropriate. A p value of <0.05 was taken to be the minimum level of statistical significance. Single Nucleotide Polymorphisms in NTCP—To identify coding region SNPs, initial screening PCR was performed on all five exons of NTCP from genomic DNA samples of 50 European Americans, 50 African Americans, and 50 Chinese Americans and analyzed by direct sequencing. Obtained sequences were compared with the published NTCP reference sequence (GenBank™ accession number NM_003049). Two nonsynonymous SNPs were identified by direct sequencing of PCR products from this initial screening of genomic DNA samples. The allelic variant C800T (Ser267 → Phe), in exon 4 of NTCP, was observed in 6 of 50 Chinese American DNA samples but was not seen in European Americans or African Americans (Fig. 1B). Another allelic variant T668C (Ile223 → Thr), in exon 3 of NTCP, was identified in 4 of 50 African American DNA samples but not seen in European Americans
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