Polymorphisms in Human Organic Anion-transporting Polypeptide 1A2 (OATP1A2)
Organic anion-transporting polypeptide 1A2 (OATP1A2) is a drug uptake transporter known for broad substrate specificity, including many drugs in clinical use. Therefore, genetic variation in SLCO1A2 may have important implications to the disposition and tissue penetration of substrate drugs. In the present study, we demonstrate OATP1A2 protein expression in human brain capillary and renal distal nephron using immunohistochemistry. We also determined the extent of single nucleotide polymorphisms in SLCO1A2 upon analyses of ethnically defined genomic DNA samples (n = 95 each for African-, Chinese-, European-, and Hispanic-Americans). We identified six nonsynonymous polymorphisms within the coding region of SLCO1A2 (T38C (I13T), A516C (E172D), G559A (A187T), A382T (N128Y), A404T (N135I), and C2003G (T668S)), the allelic frequencies of which appeared to be ethnicity-dependent. In vitro functional assessment revealed that the A516C and A404T variants had markedly reduced capacity for mediating the cellular uptake of OATP1A2 substrates, estrone 3-sulfate and two δ-opioid receptor agonists, deltorphin II, and [d-penicillamine2,5]-enkephalin. On the other hand, the G559A and C2003G variants appeared to have substrate-dependent changes in transport activity. Cell surface biotinylation and immunofluorescence confocal microscopy suggested that altered plasma membrane expression of the transporter may contribute to reduced transport activity associated with the A516C, A404T, and C2003G variants. The A404T (N135I) variant also showed a shift in the apparent molecular size, indicative of alterations in glycosylation status. Taken together, these data suggest that SLCO1A2 polymorphisms may be an important yet unrecognized contributor to inter-individual variability in drug disposition and central nervous system entry of substrate drugs. Organic anion-transporting polypeptide 1A2 (OATP1A2) is a drug uptake transporter known for broad substrate specificity, including many drugs in clinical use. Therefore, genetic variation in SLCO1A2 may have important implications to the disposition and tissue penetration of substrate drugs. In the present study, we demonstrate OATP1A2 protein expression in human brain capillary and renal distal nephron using immunohistochemistry. We also determined the extent of single nucleotide polymorphisms in SLCO1A2 upon analyses of ethnically defined genomic DNA samples (n = 95 each for African-, Chinese-, European-, and Hispanic-Americans). We identified six nonsynonymous polymorphisms within the coding region of SLCO1A2 (T38C (I13T), A516C (E172D), G559A (A187T), A382T (N128Y), A404T (N135I), and C2003G (T668S)), the allelic frequencies of which appeared to be ethnicity-dependent. In vitro functional assessment revealed that the A516C and A404T variants had markedly reduced capacity for mediating the cellular uptake of OATP1A2 substrates, estrone 3-sulfate and two δ-opioid receptor agonists, deltorphin II, and [d-penicillamine2,5]-enkephalin. On the other hand, the G559A and C2003G variants appeared to have substrate-dependent changes in transport activity. Cell surface biotinylation and immunofluorescence confocal microscopy suggested that altered plasma membrane expression of the transporter may contribute to reduced transport activity associated with the A516C, A404T, and C2003G variants. The A404T (N135I) variant also showed a shift in the apparent molecular size, indicative of alterations in glycosylation status. Taken together, these data suggest that SLCO1A2 polymorphisms may be an important yet unrecognized contributor to inter-individual variability in drug disposition and central nervous system entry of substrate drugs. During the past decade, there has been an increasing recognition of the critical interplay between drug transporters and drug-metabolizing enzymes as determinants of drug disposition and response. Indeed, the extent of targeted tissue entry for many drugs may be facilitated by drug transporters, which are often expressed in a tissue-specific manner with broad substrate specificities. Among the uptake transporters, members of the organic anion-transporting polypeptides (human, OATPs 1The abbreviations used are: OATP, organic anion-transporting polypeptide; SNP, single nucleotide polymorphism; BBB, blood-brain barrier; DPDPE, [d-penicillamine2,5]enkephalin; PBS, phosphate-buffered saline; rodents, Oatps) have been shown to be expressed in organs such as the central nervous system, liver, and intestine and mediate the cellular uptake of a large number of structurally divergent compounds (1Hagenbuch B. Meier P.J. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 653-665Crossref PubMed Scopus (845) Google Scholar). Within this family, OATP1A2 (SLCO1A2, also known as human OATP-A or OATP1) was the first human OATP to be cloned and characterized (2Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (373) Google Scholar). OATP1A2 mRNA has been detected in various tissues including the brain, liver, and kidney (2Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 3Steckelbroeck S. Nassen A. Ugele B. Ludwig M. Watzka M. Reissinger A. Clusmann H. Lutjohann D. Siekmann L. Klingmuller D. Hans V.H. J. Neurochem. 2004; 89: 403-417Crossref PubMed Scopus (61) Google Scholar). Substrates of OATP1A2 include endogenous compounds such as bile acids, steroid hormones, and their conjugates, thyroid hormones, as well as drugs including fexofenadine, ouabain, peptides (e.g. deltorphin II, [d-penicillamine2,5]enkephalin, DPDPE), and the toxin, microcystin (2Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 4Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (550) Google Scholar, 5Eckhardt U. Schroeder A. Stieger B. Hochli M. Landmann L. Tynes R. Meier P.J. Hagenbuch B. Am. J. Physiol. 1999; 276: G1037-G1042PubMed Google Scholar, 6Cvetkovic M. Leake B. Fromm M.F. Wilkinson G.R. Kim R.B. Drug Metab. Dispos. 1999; 27: 866-871PubMed Google Scholar, 7Gao B. Hagenbuch B. Kullak-Ublick G.A. Benke D. Aguzzi A. Meier P.J. J. Pharmacol. Exp. Ther. 2000; 294: 73-79PubMed Google Scholar). In humans, OATP1A2 has been reported to be the only OATP transporter detected in the brain capillary endothelium at present, suggesting that OATP1A2 may play a critical role in the central nervous system penetration of many drugs and hormones across the blood-brain barrier (BBB) (7Gao B. Hagenbuch B. Kullak-Ublick G.A. Benke D. Aguzzi A. Meier P.J. J. Pharmacol. Exp. Ther. 2000; 294: 73-79PubMed Google Scholar, 8Hagenbuch B. Gao B. Meier P.J. News Physiol. Sci. 2002; 17: 231-234PubMed Google Scholar). Given the extent of its substrate specificity and expression in organs of importance to drug disposition and response, genetic variations in SLCO1A2 may have significant pharmacologic and toxicologic consequences. In the present study, we were able to confirm the expression of OATP1A2 at the level of the brain capillaries that make up the BBB. Moreover, we describe the identification and functional characterization of SNPs in SLCO1A2 from a population of European-, Chinese-, Hispanic-, and African-Americans. Genotypic frequencies of six nonsynonymous polymorphisms within the coding region of SLCO1A2 were dependent on ethnicity, and some of the genetic variants were associated with markedly reduced uptake transport activity for estrone 3-sulfate and two δ-opioid receptor agonists, [d-penicillamine2,5] enkephalin and deltorphin II. Our data indicate that SLCO1A2 polymorphisms may contribute to inter-individual variability in drug disposition and may be a heretofore unrecognized factor governing the central nervous system entry of some drugs. Materials—Radiolabeled [3H]estrone 3-sulfate (53 Ci/mmol), [tyrosyl-3,5-3H]deltorphin II (2-d-Ala, 38.5 Ci/mmol), [tyrosyl-2,6–3H(N)]enkephalin (d-penicillamine2,5, DPDPE, 34.0 Ci/mmol), and unlabeled estrone3-sulfate were purchased from PerkinElmer Life Sciences. Unlabeled deltorphin II and DPDPE were obtained from Bachem (King of Prussia, PA). 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 and the monoclonal mouse anti-V5 antibody were purchased from Invitrogen. Genomic DNA isolated from peripheral blood lymphocytes of healthy European-, African-, Chinese-, and Hispanic-American volunteers was purchased from Coriell Cell Repositories (Camden, NJ). An enzymatic deglycosylation kit was obtained from Glyko (San Leandro, CA), and tunicamycin was purchased from Calbiochem. Immunohistochemistry reagents were obtained from Biogenex (San Ramon, CA). All other chemicals and reagents, unless stated otherwise, were obtained from Sigma and were of the highest grade available. Immunohistochemistry of Normal Human Brain, Kidney, and Liver Sections—The rabbit polyclonal antiserum against human OATP1A2 was custom-made using a synthesized C-terminal polypeptide of OATP1A2 (ECKDIYQKSTVLKDDELKTKL, Invitrogen) as the epitope. Paraffin-embedded sections from normal human brain, kidney, and liver tissues (5 μm) were obtained from Human Tissue Acquisition Shared Resources at Vanderbilt-Ingram Cancer Center. Histopathological analyses were performed on Hematoxylin and Eosin-stained sections from the paraffin tissue specimens. Sections were deparaffinized using EZ-Dewax (Biogenex) followed by the antigen retrieval procedure using Retrieve-It® (pH 8, Biogenex) in the case of brain and liver tissue sections. Sections were incubated with the peroxidase blocking reagent (Biogenex) for 10 min at room temperature to quench endogenous peroxidase activity. After rinsing with PBS, sections were incubated with blocking buffer (Powerblock, Biogenex) for 60 min and subsequently incubated with anti-OATP1A2 antiserum diluted in blocking buffer (1:100–1:500 dilutions) for 2 h at room temperature. After three washes, sections were incubated with the avidin and biotin blocking reagents (avidin-biotin blocking kit, Biogenex) to reduce nonspecific background staining due to endogenous biotin. After three washes with PBS containing 0.4% Triton X-100, sections were incubated with biotinylated anti-rabbit IgG (Biogenex) for 20 min and then with streptavidin-horseradish peroxidase conjugate (Biogenex) for 20 min. After washes, the immune reaction was visualized using 3-amino-9-ethylcarbazole (Biogenex), and nuclei were counterstained with hematoxylin (Biogenex). The specificity of immunoreactive signals for OATP1A2 was verified by various negative controls, which were incubated with the rabbit preimmune serum, blocking buffer, or polyclonal antiserum that had been neutralized by preincubation with the antigenic peptide at 37 °C for 2 h. Identification of SNPs in SLCO1A2 and Determination of Genotypic Frequencies—Initially, total genomic DNA was isolated from blood samples obtained from healthy volunteer subjects (46 European- and 32 African-Americans) residing in middle-Tennessee who were judged to be healthy on the basis of medical history, physical examination, and laboratory test indicative of normal cardiac, renal, and liver function. The protocol was approved by the Vanderbilt University Institutional Review Board, and informed consent was obtained. Further analyses of genetic variations in the human SLCO1A2 gene used ethnically defined genomic DNA samples from healthy volunteers (European-, African-, Chinese-, and Hispanic-American, n = 95 each). Initial screening involved the amplification of the 14 exonic regions of SLCO1A2 followed by nonisotopic single-strand conformational polymorphism analysis. PCR was typically carried out at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s for 30 cycles using 25 ng of genomic DNA previously digested with the restriction endonuclease XhoI (New England Biolabs, Inc., Beverly, MA). Variations in the single-strand mobility patterns were clearly visualized by ethidium bromide staining. A search of available SNP databases, including PharmGKB (www.pharmgkb.org), IMS-JST Japanese SNP (snp.ims.u-tokyo.ac.jp), NCBI dbSNP (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp), and GeneCards (bioinfo.weizmann.ac.il/cards), was performed to identify any additional polymorphisms. The genotypic frequencies of the nonsynonymous SNPs identified by single-strand conformational polymorphism and available data base analyses (T38C, A516C, G559A, A382T, A404T and C2003G) were determined by either direct sequencing (ABI 3700 DNA Analyzer, Applied Biosystems Inc., Foster City, CA) or temperature-dependent capillary electrophoresis (Reveal®, SpectruMedix, State College, PA) of PCR products. Generation of Wild-type and Variant SLCO1A2 SLCO1A2 containing the in the vector was a gift from Dr. Meier The of human SLCO1A2 was by PCR using as the and the pEF6/V5-His-TOPO® vector The PCR and were to the SLCO1A2 the to the pEF6/V5-His-TOPO® and with the SLCO1A2 in the from the T7 region were verified by direct sequencing and to the number was and the of the first base of the was as using the kit CA) was performed to the identified nonsynonymous allelic A516C G559A A382T A404T and C2003G The of each was of Variant SLCO1A2 transport in were with vaccinia at a of of 10 in and to for 30 min at 37 in each well were then with of or variant SLCO1A2 either pEF6/V5-His-TOPO® or vector with and incubated at 37 °C for h. The any was used as vector was then using drug as previously R.B. Leake B. M. J. A. Wilkinson G.R. J. Pharmacol. Exp. Ther. 1999; Google Scholar). estrone deltorphin II, and DPDPE transport drug uptake the first was in the of of unlabeled was determined by out using the DNA the transporter and this was then from the total uptake in the of the transporter was carried out to of the uptake and the at which the uptake CA). All were carried out in on at OATP1A2 Cell were on and with or variant SLCO1A2 pEF6/V5-His-TOPO® using a protocol for transport were with and then with a at °C for h. the were three with containing and then incubated for 20 min at °C with the buffer to the After with were with of buffer Triton X-100, containing Applied at °C for h with of was to of and incubated for h at room temperature. were with buffer, and the biotinylated were from the by buffer and for 10 min at 95 to the biotinylated the total were to for of OATP1A2 with monoclonal anti-V5 antibody Invitrogen) and the protein with polyclonal antibody as previously Leake Kim R.B. J. 276: Full Text Full Text PDF PubMed Scopus Google Scholar). and were on and at with 2 of or variant SLCO1A2 using After were for 10 min in were then for 10 min in PBS containing Triton After rinsing with PBS, were in blocking buffer in for h at room temperature. were then incubated with monoclonal antibody against in blocking for 2 h at room temperature. After three washes in PBS containing were incubated with antibody with the for 30 min at 37 During the washes in PBS containing was for staining. microscopy was performed with a with a A was used for were obtained using single and with the any were used as with with antibody were also used as an additional confocal were obtained by either or across the and were performed with and of and Cell were on and with and using a protocol to that for the transport and OATP1A2 variants and were isolated as previously and to enzymatic deglycosylation to the provided by the Leandro, CA). total or OATP1A2 protein samples were to and enzymatic deglycosylation by and were by and by for of immunoreactive with monoclonal anti-V5 antibody In an of the glycosylation of was to a of a and OATP1A2 protein samples were isolated and by as of the between various was performed using of of were to OATP1A2 staining of OATP1A2 was performed to the tissue of OATP1A2 in the human brain and kidney using rabbit polyclonal antiserum against human of the antiserum were used to the of expression in background staining was staining was performed with the highest antiserum In normal human brain detected OATP1A2 expression in capillary of the polyclonal antiserum against OATP1A2 with the antigenic peptide staining in brain capillary OATP1A2 expression in human kidney was to the of distal In human liver OATP1A2 expression appeared to be to the Variations in Human SLCO1A2 single-strand conformational polymorphism analyses of 14 of SLCO1A2 genomic DNA samples from European- and 32 African-Americans) and a search of available SNP identified nonsynonymous on the reported genotypic frequencies and of we determined the allelic frequencies of six nonsynonymous SNPs (T38C, A516C, G559A, A382T, A404T, and C2003G) using ethnically defined genomic DNA (European-, African-, Chinese-, and Hispanic-American, n = 95 and A516C polymorphisms were in and and these SNPs were with G559A variation was only in A382T was only in A404T polymorphism was detected in genomic DNA samples used in the present C2003G variation was in and with allelic frequencies of and of the six nonsynonymous SNPs were in frequencies of nonsynonymous SNPs in OATP1A2 various were from African-, European-, Chinese-, and n = 95 and dbSNP and and in a of SLCO1A2 Variant of expression SLCO1A2 allelic variants was using the vaccinia system was of known substrates, such as estrone 3-sulfate deltorphin II and DPDPE the transport of the OATP1A2 we the total cellular uptake μm) at 30 min. associated with and variants were the for three The variant markedly reduced transport activity estrone 3-sulfate deltorphin II and In the variant appeared to have the reduced cellular uptake of deltorphin II and DPDPE normal uptake activity for estrone a uptake uptake data the of estrone 3-sulfate and deltorphin II were for allelic variants and with screening some of the OATP1A2 variants A516C and for estrone 3-sulfate and deltorphin II the On the other hand, the variant showed altered and only for estrone 3-sulfate for deltorphin II. The other and also appeared to have altered with deltorphin II. to was to for of the OATP1A2 variants for deltorphin II using of are expressed as (n = were obtained using the of deltorphin II uptake the and Cell of OATP1A2 analyses of total OATP1A2 protein expression that the reduced transport activity of some OATP1A2 variants A404T and C2003G) may be associated with alterations in total protein In to the to the molecular of (2Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (373) Google the showed of various glycosylation by the as well as by the the variant also a shift in the mobility of immunoreactive indicative of altered glycosylation We also in surface expression for the transport capacity of variant OATP1A2 Cell surface biotinylation were performed to plasma OATP1A2 to that with total and variants showed expression of immunoreactive and a of of and was The of surface within the biotinylated was by the of in the samples In the variant showed altered mobility the immunoreactive indicative of changes in glycosylation status. of and Cell expressed in the total and OATP1A2 protein appeared to molecular including the as well as molecular of and A and We performed enzymatic deglycosylation to this apparent various the glycosylation of total and In we also the deglycosylation of the variant showed altered mobility in with that of to enzymatic total and of and showed a significant shift in in apparent molecular are with the of additional in OATP1A2 protein as by the and were including at the of and as well as the to the deglycosylation were also carried out using an of to enzymatic deglycosylation tunicamycin the molecular of the immunoreactive of total and and protein to a extent as that with samples confirm that is a glycosylation in of OATP1A2 surface biotinylation and of total in suggested that the reduced transport of OATP1A2 variants may be due to altered of the protein to the we performed immunofluorescence confocal microscopy from the as well as with the SLCO1A2 variants antibody against and antibody with we that protein was targeted to the plasma membrane of and and The expression patterns of OATP1A2 including and suggested alterations in surface in and Drug and targeted tissue penetration are to be dependent on drug uptake and transporters R.B. Eur. J. PubMed Scopus Google Scholar, Pharmacol. PubMed Scopus Google Scholar). the extent of in of transporters such as is only has there been the of the and importance of drug uptake transporters to the drug disposition A of to as OATP, to have to drug expression of OATPs in organs such as the liver, kidney, and brain to the extent of drug and central nervous system a and system has been to an and identification of OATP and gene (1Hagenbuch B. Meier P.J. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 653-665Crossref PubMed Scopus (845) Google Scholar). a of OATP transporters at the molecular level is to the of drugs with and In this we describe the expression of OATP1A2 in human brain and kidney and the identification and functional characterization of nonsynonymous SNPs in We first the expression of OATP1A2 using human tissues immunohistochemistry. We an antibody against the C-terminal of which appeared to be with against and In the liver, OATP1A2 expression was to in In the kidney, OATP1A2 was expressed on the of distal with the reported (7Gao B. Hagenbuch B. Kullak-Ublick G.A. Benke D. Aguzzi A. Meier P.J. J. Pharmacol. Exp. Ther. 2000; 294: 73-79PubMed Google we detected OATP1A2 in the brain Genotypic analyses of subjects from various revealed that the allelic frequencies of six identified SNPs in SLCO1A2 are dependent on the extent of genetic variation appeared with other members of OATPs such as and Leake Kim R.B. J. 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. A. J. Pharmacol. Exp. Ther. 2002; PubMed Scopus Google Scholar). the transport of the and six SLCO1A2 allelic variants was in the and variants markedly reduced transport activity three estrone deltorphin II, and DPDPE OATP1A2 variants showed altered transport activity in a manner and reduced uptake transport activity with estrone 3-sulfate with deltorphin II and the was to be the case with and of transport was that at and have functional consequences. An OATP1A2 membrane on suggested that associated with and are in the and the to be in regions in with other OATP regions have been suggested to have a important in of substrate specificity on with an cloned from liver Am. J. Physiol. 2002; PubMed Scopus Google Scholar). transport of these OATP1A2 variants and may in to reduced total OATP1A2 protein In the variant reduced expression in and using surface biotinylation or immunofluorescence confocal confocal microscopy revealed significant of expression for and be that there may be a of in the extent of surface expression for variants such as for the there appeared to be in of in surface expression between the two In for of at this appeared to in altered glycosylation as by a shift in molecular on Indeed, the A404T (N135I) in the is at a for The of has been to play a number of important including of of protein and of protein A that also an important role in transport activity J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). tissue expression and genetic variation in OATP1A2 are together, a number of or drug entry the of OATP1A2 in the distal of the a important role for this transporter to of drugs that are or at the level of the expression at the level of the is of clinical the broad substrate specificity for this including peptide such as deltorphin II and DPDPE The BBB, by the of brain capillary a number of transporter on the and of transporters has been shown to be critical to the and entry of compounds the brain 2004; PubMed Scopus Google Scholar). Among transporters expressed at the BBB, OATP1A2 may be of the uptake transporters that drugs may to the to the brain (7Gao B. Hagenbuch B. Kullak-Ublick G.A. Benke D. Aguzzi A. Meier P.J. J. Pharmacol. Exp. Ther. 2000; 294: 73-79PubMed Google Scholar). The of and compounds may on transport across membrane to their cellular in the central nervous genetic variations associated with OATP1A2 may only the disposition of endogenous and compounds in organs such as the kidney also the extent of drug to tissue such as the brain, to inter-individual variability in drug In we the identification and functional characterization of polymorphisms in human this the first of expression and characterization of polymorphisms in human OATP1A2 and the for of the of SLCO1A2 polymorphisms in its substrate specificity and expression in organs such as the brain and kidney, genetic variations in SLCO1A2 may be an important contributor to inter-individual variability in drug disposition and central nervous system entry of substrate drugs. were performed in the of the Vanderbilt University Cell by Institutes of and