Functional Complementation between a Novel Mammalian Polygenic Transport Complex and an Evolutionarily Ancient Organic Solute Transporter, OSTα-OSTβ

Abstract

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTα and OSTβ or mouse OSTα and OSTβ. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostα-Ostβ proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25–41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTα proteins are predicted to contain seven transmembrane helices, whereas the OSTβ sequences are predicted to have a single transmembrane helix. Human OSTα-OSTβ and mouse Ostα-Ostβ cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTα-OSTβ combinations (i.e. OSTα-OSTβ pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTα-OSTβ also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17β-d-glucuronide or p-aminohippurate. OSTα and OSTβ were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTα and OSTβ mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland. These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTα and OSTβ or mouse OSTα and OSTβ. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostα-Ostβ proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25–41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTα proteins are predicted to contain seven transmembrane helices, whereas the OSTβ sequences are predicted to have a single transmembrane helix. Human OSTα-OSTβ and mouse Ostα-Ostβ cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTα-OSTβ combinations (i.e. OSTα-OSTβ pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTα-OSTβ also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17β-d-glucuronide or p-aminohippurate. OSTα and OSTβ were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTα and OSTβ mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland. Cellular homeostasis requires the regulated entry and exit of a multitude of compounds across the plasma membrane. Cells must take up specific amounts of nutrients, metabolic precursors, inorganic ions, signaling molecules, and other macromolecules while also exporting signaling molecules, hormones, electrolytes, metabolic waste products, and xenobiotics. Recent studies have described some of the genes involved in these transport processes; however, it is clear that many other genes and gene products remain to be identified and characterized (1Saier M.H. Microbiol. Mol. Biol. Rev. 2000; 64: 354-411Crossref PubMed Scopus (679) Google Scholar, 2Borst P. Evers R. Kool M. Wijnholds J. J. Natl. Cancer Inst. 2000; 92: 1295-1302Crossref PubMed Scopus (1517) Google Scholar, 3Keppler D. Konig J. Semin. Liver Dis. 2000; 20: 265-272Crossref PubMed Scopus (211) Google Scholar, 4Kullak-Ublick G.A. Stieger B. Hagenbuch B. Meier P.J. Semin. Liver Dis. 2000; 20: 273-292Crossref PubMed Scopus (233) Google Scholar, 5Suzuki H. Sugiyama Y. Semin. Liver Dis. 2000; 20: 251-263Crossref PubMed Scopus (77) Google Scholar, 6Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10387) Google Scholar, 7Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. et al.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17336) Google Scholar). Using a comparative approach, a novel type of organic solute and steroid transporter was recently identified in the liver of an evolutionarily ancient vertebrate, the little skate Raja erinacea (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). In contrast to all other organic anion carriers, this skate transporter is generated by co-expression of two distinct and novel gene products, Ostα and Ostβ. 1The abbreviations used are: Ost/OST, organic solute transporter; hOSTα, human OSTα; mOSTα, mouse OSTα; sOstα, skate Ostα; ORF, open reading frame; TM, transmembrane; PBS, phosphate-buffered saline; RT-PCR, reverse transcription PCR; SNAP, soluble N-ethylmaleimide-sensitive fusion attachment protein; SNARE, SNAP-25 receptor.1The abbreviations used are: Ost/OST, organic solute transporter; hOSTα, human OSTα; mOSTα, mouse OSTα; sOstα, skate Ostα; ORF, open reading frame; TM, transmembrane; PBS, phosphate-buffered saline; RT-PCR, reverse transcription PCR; SNAP, soluble N-ethylmaleimide-sensitive fusion attachment protein; SNARE, SNAP-25 receptor. Substrates for this multispecific transporter include estrone 3-sulfate, taurocholate, digoxin, and prostaglandin E2. Interestingly, the overall predicted membrane topology of skate Ostα-Ostβ is similar to that of the heterodimeric sensory rhodopsins, suggesting that Ostα-Ostβ may have evolved from an ancestral rhodopsin-like molecule but has acquired the ability to transport steroids and eicosanoids, compounds that also function as ligands for some G-protein-coupled receptors. Initially, Ostα and Ostβ orthologues were not identified in the human genome or in any other sequenced genomes, indicating that these genes might be specific to marine elasmobranchs. However, sequences for hypothetical human and mouse proteins have recently been entered into the data bases that exhibit 25–41% predicted amino acid sequence identity with skate Ostα and Ostβ (see “Results”). The present study tested whether these mammalian genes are expressed, whether they encode for orthologues of the skate gene products, and if so, whether they functionally complement the transport activity of one another. Materials and Animals—[G-3H]Taurocholic acid (3.47 Ci/mmol), [3H]estrone 3-sulfate (53 Ci/mmol), [G-3H]digoxin (19 Ci/mmol), [3H]prostaglandin E2 (200 Ci/mmol), [3H]estradiol 17β-d-glucuronide (50 Ci/mmol), and p-[glycyl-2-3H]aminohippuric acid (4.08 Ci/mmol) were purchased from PerkinElmer Life Sciences. Chemicals were obtained from Sigma or J. T. Baker Inc. Molecular biology reagents were purchased from Invitrogen; Clontech, Palo Alto, CA; Integrated DNA Technologies, Coralville, IA; Qiagen, Valencia, CA; Origene, Rockville, MD; Ambion, Austin, TX; and Promega, Madison, WI. Mature Xenopus laevis were purchased from Nasco, Fort Atkinson, WI. Animals were maintained under a constant light cycle at a room temperature of 18 °C. Cloning Strategy—A multistep RT-PCR-based strategy was employed to obtain cDNAs for the putative open reading frames (ORFs) of hypothetical proteins OSTα (GenBank™/EBI accession number CAC51162), OSTβ (GenBank™/EBI accession number XP_058693), and mOSTβ (GenBank™/EBI accession number XP_134984/AY279396). Oligonucleotide primers were designed to generate ORFs for each gene based on the cDNA sequences listed on GenBank™ (Table I). RT-PCR products of predicted sizes were created for putative hOSTα, hOSTβ, and mOstβ using either human or mouse liver poly(A)+ RNA as a template (Clontech). The reaction products were isolated via agarose gel electrophoresis and ligated into the pCR-II TOPO vector (Invitrogen) utilizing Invitrogen's TOPO TA cloning kit. The resulting plasmids were used to transform DH5α Escherichia coli, from which positive clones were selected for ampicillin resistance and blue/white staining. Upon isolation and identification, a single clone expressing the new plasmid was used as a template in a PCR with primers designed to generate a final product flanked by two unique restriction sites (Table I). Following isolation and enzymatic digestion, the cDNAs with sticky ends were directionally ligated into the pSP64 poly(A) vector. These plasmids then were used to transform DH5α E. coli, and positive clones were selected for ampicillin resistance. Plasmid purifications were completed using Promega's DNA Wizard kit.Table IPrimer sequences used to clone and quantify human and mouse Ostα-Ostβ The cDNA for the hypothetical mOstα (GenBank™/EBI accession numbers BC024441.1 and AAH25912) was obtained from the American Type Culture Collection (ATCC #6994476). This clone was used as a template in a PCR to generate the ORF for GenBank™ sequence BC024441.1. The primers were designed to add a HindIII site to the 5′ end and an XbaI site to the 3′ end of the PCR product (Table I). The PCR product was purified by agarose gel electrophoresis, and the band of expected size (1060 bp) was excised and enzymatically digested with HindIII and XbaI. The resulting cDNA sequence was then directionally cloned into the pSP64 poly(A) vector and used to transform DH5α E. coli. A positive clone was selected, and the plasmid was isolated with Promega's DNA Wizard miniprep kit. Final clones for all four genes were sequenced in both directions with a series of specific oligonucleotide primers at the Mount Desert Island Biological Laboratory DNA Sequencing Core (Salsbury Cove, ME) to confirm sequence identity. The plasmids were then linearized with EcoRI and used to synthesize cRNA via Ambion's mMessage mMachine kit for injection into Xenopus oocytes. Real Time Quantitative PCR to Determine Tissue and of and in Human Tissue cDNAs were purchased from oligonucleotide primers were designed to of hOSTα, hOSTβ, and human for in PCR (Table I). were and on a light from RT-PCR kit was used for PCR levels are as a to each Xenopus and Transport of Xenopus was as described by PubMed Scopus Google and employed in N. W. Li L. J. Google Scholar, L. Meier P.J. Ballatori N. J. Biol. PubMed Scopus Google Scholar, L. Meier P.J. Ballatori N. Mol. 2000; PubMed Scopus Google Scholar). and were selected and at 18 in and with and The oocyte was and oocytes, as with a and a distinct were selected for Transport studies were as described N. W. Li L. J. Google Scholar, L. Meier P.J. Ballatori N. J. Biol. PubMed Scopus Google Scholar). DNA and cDNA clones were sequenced in both directions at the Mount Desert Island Biological Laboratory DNA Sequencing The sequences for all clones were obtained using oligonucleotide was with the DNA and sequence from Inc. topology and putative were by and by J. J. Mol. Biol. PubMed Scopus Google Scholar). The of signal was with the of and Human OSTα and of and Human was to the 3′ end of skate and human OSTα and the was to the 3′ end of skate and human OSTβ were created via PCR using skate and human OSTα and OSTβ cDNA as with oligonucleotide primers the or sequences flanked by unique restriction PCR products of predicted size were identified by agarose gel electrophoresis, and purified with agarose gel kit. The isolated were into the pSP64 poly(A) and that vector was used to transform DH5α E. coli. clones were and plasmid DNA isolated using Promega's DNA Wizard plasmid isolation kit. restriction and to clone cRNA was for each of the four using Ambion's mMessage mMachine kit. of Xenopus on cRNA injection were in for on by four of each at room temperature and an at in and were at room temperature with for with by a in in were then with for with by an at °C. The were with either with or with for were in for two for and then The with the were with an of in the for was by the with for two for and then Cells were using a on a data from measuring of were to the by and with were from these of data measuring of of in the and of was by and to Cloning Human OSTα and OSTβ and Ostα and human and mouse proteins (GenBank™/EBI accession numbers and that were recently to the data bases exhibit predicted amino acid identity with skate Ostα and amino acid identity with each other of the many amino acid the of amino acid is skate Ostα and these putative mammalian orthologues and the hypothetical mouse and human proteins Interestingly, all amino acid sequences a highly of in a predicted transmembrane and This overall amino acid identity with the in the human, mouse, and skate gene products that they are human and mouse orthologues of skate Ostβ were recently entered into the data but these sequences exhibit only predicted amino acid identity with skate Ostβ The hypothetical human with GenBank™ sequence and the hypothetical mouse with GenBank™ sequence exhibit and predicted amino acid identity with skate These hypothetical human and mouse proteins exhibit amino acid identity with each both for proteins amino with a putative single RT-PCR-based strategy was employed to obtain cDNAs for the predicted ORFs of putative human OSTα and OSTβ using human liver and of putative mouse Ostβ using mouse liver The cDNA for the putative mouse Ostα was obtained from the American Type Culture Collection (ATCC #6994476). of the cDNAs for the four genes that both mouse clones were to GenBank™ whereas the human sequences by a single in the reading of each in the human gene sequences in amino acid and accession numbers and is whether the are to or whether they from are to be sequence as both in of sequence with the human DNA data human OSTα sequence whereas the GenBank™ cDNA sequence for the with accession number an at the in a of the This a at of human OSTα in whereas the GenBank™ sequence an In human OSTβ sequence from the DNA sequence at the in a at of the ORF of an a the sequence for human OSTβ obtained in the present study a at in whereas the GenBank™ sequence a the human liver poly(A)+ RNA used as a template for RT-PCR in the present study was from four the reaction product the used for cloning human OSTβ was Upon it was that an of and at this not indicating that this may be a A of the human data that human OSTα is on and is by nine in the whereas human OSTβ is on and is by four in the Ostα is on and is by nine at whereas mouse Ostβ is on and is by four at The Human and as whether the human and mouse proteins function as organic solute of [3H]estrone 3-sulfate was in laevis with cRNA from the human, mouse, or skate co-expression of skate Ostα and Ostβ was required to generate transport activity (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). the putative human OSTα and mouse OSTα were expressed individually in oocytes, transport activity was however, when these proteins were with skate a transport signal was obtained indicating that human OSTα and mouse OSTα functionally complement the corresponding skate human OSTβ and mouse OSTβ not transport activity when expressed individually in but generated a transporter when with skate Ostα the human and mouse proteins not only are orthologues of the skate proteins but also are able to complement each other across co-expression of the two human or the two mouse proteins generated a transport as the pairs of Human and with human OSTα and OSTβ cRNA or with mouse Ostα and Ostβ cRNA were able to transport taurocholate, estrone 3-sulfate, digoxin, and prostaglandin E2 but not estradiol 17β-d-glucuronide or indicating that this transport is multispecific and that it may in of steroids and This is similar to that of skate Ostα-Ostβ (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). The skate, mouse, and human a number of other as Transport was and inhibited by and other organic (Table of the in the oocyte with either or on estrone 3-sulfate indicating that transport is not to the of estrone 3-sulfate into human or mouse were saturable, the were and The for estrone 3-sulfate by skate Ostα-Ostβ is (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). of [3H]estrone 3-sulfate in and was inhibited by a of and other organic (Table for skate Ostα-Ostβ (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google [3H]estrone 3-sulfate was inhibited by acid and acid (Table of [3H]estrone 3-sulfate in expressing human OSTα-OSTβ or mouse OSTα-OSTβ were with of cRNA of cRNA or of mOstα cRNA of mOstβ cRNA were with [3H]estrone 3-sulfate of and and was for at °C. of the are of in distinct oocyte each in of and anionic on human OSTα-OSTβ and mouse transport of [3H]estrone 3-sulfate acid from from from from from from from acid from from 3-sulfate, from from from from from from from from from from from from from from in a new Tissue of Human OSTα and OSTβ OSTα and OSTβ levels were in human cDNAs and were expressed to levels using PCR The results that OSTα and OSTβ are widely expressed in human that levels of OSTα also levels of OSTβ mRNA, indicating of these levels of both mRNAs were in testis, colon, liver, small intestine, kidney, ovary, and adrenal and levels were in and The for OSTα and OSTβ was of in and not of OSTα and OSTβ to the into the by which OSTα and OSTβ to generate a were and these were tested for activity and in Xenopus oocytes. The was to the 3′ end of skate and human OSTα and the was to the 3′ end of skate and human OSTβ was transport activity when the proteins were expressed in oocytes; however, when human or skate and were co-expressed, was estrone 3-sulfate transport activity Interestingly, that and were able to reach the plasma membrane when expressed either individually or in pairs the of the proteins to generate transport activity not to be to to the of skate Ostα-Ostβ or human OSTα-OSTβ in Xenopus oocytes. The was to skate Ostα and human and the was to skate Ostβ and human OSTβ cRNA from these four was into either individually and skate and human and skate and and human or as pairs for each and for skate and and for were either with an and by an of and for and for or with a and for and for The present results identify a novel mammalian organic solute transporter for which the is widely distributed and highly expressed in human This transporter is unique mammalian organic anion in that it is generated when two distinct gene products are co-expressed, namely a putative membrane and a OSTβ. Interestingly, the human and mouse proteins were able to complement each other as as from an evolutionarily ancient vertebrate, the little skate, indicating a of the of this transporter are not and ability to transport steroids and prostaglandin E2 an in of OSTα and OSTβ was by the cloning of genes from the liver of an evolutionarily ancient marine vertebrate, the little skate (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). when skate Ostα and Ostβ genes were identified in genes were not present in any of the sequenced genomes, the human suggesting that they may be unique to (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). However, the human genome in many genes have not been human and mouse genes that recently were entered into the data bases are predicted to encode proteins 25–41% amino acid identity with skate Ostα and Ostβ and this of amino acid identity is it is not an of and the of amino acid these skate and mammalian proteins may be the this the present study whether these hypothetical human and mouse genes are expressed in human and mouse liver, and if so, whether they function as organic solute results demonstrate not only that human OSTα-OSTβ and mouse Ostα-Ostβ are orthologues of skate Ostα-Ostβ but also that these proteins are able to functionally complement one across the OSTα proteins from or are able to with any of the OSTβ proteins from these to generate a a of The by which OSTα and OSTβ to generate transport activity is the present results that co-expression is not required for to the OSTα and OSTβ were able to reach the plasma membrane when expressed individually in oocytes; however, they were not These a function and that either one may a or the two proteins may be a or The present results also that levels of transport activity are generated by proteins that exhibit only amino acid suggesting that only a amino may be required for this human OSTα was by human OSTβ or skate Ostβ only a amino acid identity for the latter two proteins the OSTα and OSTβ may be by their or by amino acid sequences or may the of a as or Interestingly, the predicted transmembrane of human, mouse, and skate OSTα are as are of the OSTβ proteins and that these proteins the of the predicted and in OSTα and OSTβ from human, mouse, and skate are similar in and the and The only is skate which has a amino however, the amino of skate Ostβ are predicted to be a signal (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google that the may be in to the human and mouse proteins. The membrane these evolutionarily divergent proteins that this membrane is for the amino acid identity the human, mouse, and skate proteins is not to the but is also in putative and and amino acid highly in the an of that in a predicted and The of this in OSTα is not it may function either as a or a site of with or as a site of membrane In in and that contain include the human (GenBank™/EBI accession number a (GenBank™/EBI accession number a gene for human (GenBank™/EBI accession number the two proteins of function and a putative human transporter (GenBank™/EBI accession number and a (GenBank™/EBI accession number each of these proteins a they sequence identity with is to however, that many of these proteins are with the membrane and are either or involved in membrane transport Biol. PubMed Google Scholar, J.C. Biol. PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar, J. C. C. P. P. A. P. PubMed Google Scholar, P. P. PubMed Scopus Google Scholar, J. N. P. 2001; PubMed Scopus Google Scholar). is also to that human, mouse, and skate OSTα and OSTβ proteins all to have sequences in their putative Ostα and Ostβ and mouse OSTβ have an whereas human OSTα and mouse OSTα have an sequence at the corresponding in the sequence and sequences in proteins function as or that must be the corresponding be from the L. M. A. PubMed Scopus Google Scholar, N. B. PubMed Scopus Google Scholar, M. 2000; PubMed Scopus Google Scholar). are for OSTα and OSTβ in the mouse or human genomes, the OSTβ sequences from mouse and skate exhibit a of predicted amino acid identity with the amino (i.e. the and the of a that to the (8Wang W. Seward D.J. Li L. Boyer J.L. Ballatori N. Proc. Natl. Acad. Sci. 2001; 98: 9431-9436Crossref PubMed Scopus (100) Google Scholar). are involved in and and one study has identified a for these proteins in organic solute transport J.R. D. et al.Science. PubMed Scopus Google Scholar). and J.R. D. et al.Science. PubMed Scopus Google a that was with the of transport activity by has only one putative these that this may be one of a or may to a these have not been these by and that some of the of proteins or as OSTβ may to membrane transport activity. However, of on sequence these identify or that exhibit only a of sequence identity. and may be identified that are not from sequence The of for OSTα and OSTβ and the that this transporter has for the that these genes a and unique in The present results that OSTα-OSTβ function as a transporter for steroids as estrone 3-sulfate, taurocholate, and digoxin, as as the prostaglandin E2 steroids and are involved in many this transporter may a in these one of OSTα-OSTβ is to entry exit of signaling for an of these genes is by the of OSTα and OSTβ and by the levels of in human was in of with levels in testis, colon, liver, small intestine, kidney, ovary, and adrenal gland. In these tissues, was present at of the levels of is a this that OSTα and OSTβ are expressed at However, not with studies are to as as the and of these proteins.