©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Primary Structure and Functional Expression of a cDNA Encoding the Bile Canalicular, Purine-specific Na-Nucleoside Cotransporter (*)

Mingxin Che , Daniel F. Ortiz , Irwin M. Arias

From the (1) Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We previously characterized a purine-specific Na-nucleoside cotransport system in bile canalicular membrane. The function of this transport system may be related to conserving nucleosides and preventing cholestasis. We report here the isolation of a cDNA encoding a Na-dependent nucleoside transporter from rat liver using an expression cloning strategy. The substrate specificities and kinetic characteristics of the cloned cotransporter are consistent with the properties of the Na-dependent, purine-selective nucleoside transporter in bile canalicular membranes. The nucleotide sequence predicts a protein of 659 amino acids (72 kDa) with 14 putative membrane-spanning domains. Northern blot analysis showed that the transcripts are present in liver and several other tissues. Data base searches indicate significant sequence similarity to the pyrimidine-selective nucleoside transporter (cNT1) of rat jejunum. Although these two subtypes of Na-nucleoside cotransporter have different substrate specificities and tissue localizations, they are members of a single gene family.


INTRODUCTION

Animal liver contains abundant 5-phosphoribosyl-1-pyrophosphate amidotransferase, the first enzyme in de novo purine biosynthesis, and rapidly incorporates [C]formate into purine nucleotides in vivo and in vitro(1) . Incorporation of [C]formate into bone marrow DNA purines is depressed following partial hepatectomy, which suggests that the liver is a major source of purine for salvage pathways (1) . A Na-dependent purine nucleoside cotransporter system has been localized to the bile canalicular membrane (2) . One function of this transporter is related to maintaining the nucleoside concentration in hepatocytes and preventing nucleoside loss in bile.

A major source of intracanalicular purine nucleosides is likely to be the breakdown of nucleotides by ectonucleotidases, which co-localize in bile canalicular membrane with the nucleoside transporter (3) . Both ectonucleotidases and the Na-dependent purine nucleoside transport may be important not only for purine salvage but also in regulating the effect of ATP and adenosine on liver function. ATP and adenosine exert multiple receptor-mediated effects on isolated hepatocytes (4, 5) , and it is unclear whether these purinergic receptors are canalicular. Hypoxia releases ATP from human erythrocytes and cardiac myocytes (6, 7) and produces canalicular cholestasis (8) , suggesting that intracanalicular ATP and/or its breakdown product, adenosine, may be increased in hypoxia resulting in cholestasis through increased purinergic receptor-mediated intracellular cAMP and Ca. The ectonucleotidases, purinergic receptors, and nucleoside transporter may interact coordinately to conserve nucleoside and prevent cholestasis. The cloning and expression of the canalicular Na-dependent purine nucleoside transport permits study of its regulation and relation to ectonucleotidases and purinergic receptors.

Nucleoside transport has become the focus of much experimental work because of clinical use of nucleoside analogues (9, 10) . Based on substrate specificities, Na-dependent nucleoside transporters have been classified into three subtypes: Cif (selective for purine nucleosides and uridine) (2, 11, 12) ; Cit (selective for pyrimidine nucleosides and adenosine) (13, 14) ; and Cib (broadly specific for purine and pyrimidine nucleosides) (15) . It is not known whether these transporters belong to a single structural family. Recently, two cDNA clones encoding putative Na-dependent nucleoside transporter proteins were identified. One clone was isolated from rat jejunum and encoded a nucleoside transporter with characteristics of the pyrimidine selective (Cit) nucleoside transporter (cNT1). The transcript of cNT1 was detected only in jejunum and kidney (16) . The other clone (SNST1) was isolated from rabbit kidney by hybridization with the rabbit Na-glucose cotransporter and encoded a nucleoside transporter with broad specificity for purines and pyrimidines. The transcript of SNST1 was detected only in kidney and heart (17) . The clones had no significant sequence homology. Both transcripts were absent from liver and had different substrate specificities when compared with the canalicular nucleoside transport, suggesting that the Na-dependent purine nucleoside transporter (Cif) in rat liver is a distinct gene product with unique structure and function.

The goal of this study was to isolate the cDNA encoding the Na-dependent purine nucleoside transporter (SPNT)() from rat liver by expression cloning in Xenopus laevis oocytes and determine whether SPNT and cNT1 or SNST1 belong to the same family and are evolved from a common evolutionary precursor. Based upon molecular characteristics of these functionally distinct nucleoside transporters, we are now able to study substrate binding sites, tissue-specific function, and regulation, which will provide important information for drug design and clinical application.


EXPERIMENTAL PROCEDURES

RNA Isolation and Poly(A)RNA Selection and Fractionation

Total RNA was extracted from male Wistar rat liver and other tissues using the acid guanidine thiocyanate/phenol/chloroform extraction method (18) . Poly(A) RNA was selected by oligo(dT)-cellulose (Collaborative Research Inc.) chromatography and fractionated according to size using sucrose gradient centrifugation (19) .

X. laevis Oocytes and Na-dependent Nucleoside Transport Measurements

Oocytes were dissected from ovarian fragments of X. laevis, defolliculated manually, and incubated in modified Barth's solution consisting of 73 mM NaCl, 1 mM MgCl, 0.5 mM CaCl, 25 mM HEPES, 1 mM KCl, 100 µg/ml bovine serum albumin, 10 mM NaHCO, and 50 µg/ml gentamicin at 18 °C. Nucleoside uptake into oocytes was measured by a radiotracer technique (20) . In brief, oocytes were washed with a Na-free buffer (100 mM choline chloride, 2 mM KCl, 1 mM CaCl, 1 mM MgCl, and 10 mM HEPES-Tris (pH 7.5)) and then placed in a Na (100 mM NaCl) or sodium-free (100 mM choline chloride) buffer containing [2,8-H]adenosine (32.9 Ci/mmol, DuPont NEN). After incubation for 1-120 min at 20 °C, adenosine uptake was terminated by removing the incubation medium and washing the oocytes with two 10-ml aliquots of ice-cold choline chloride buffer containing 1 mM unlabeled adenosine. Each oocyte was dissolved in 0.5 ml of 10% (w/w) SDS. After addition of 5 ml of scintillation fluid, oocyte-associated radioactivity was counted in a liquid scintillation spectrometer (Beckman, LS 1801).

Library Construction and Clone Isolation

A directional cDNA library was constructed in the plasmid vector pSPORT1 (SuperScript, Life Technologies, Inc.) using the size-fractionated poly(A) RNA that gave rise to peak Na-dependent adenosine uptake. cDNA of 2-4 kb was ligated into pSPORT1 plasmid, which was electroporated into DH cells. cRNA was transcribed in vitro using T RNA polymerase in the presence of Cap analog (mGpppG; Pharmacia Biotech Inc.) from pools of clones and injected into oocytes. A pool that induced Na-dependent adenosine uptake was progressively subdivided until a single clone with a cDNA insert of 2.9 kb was isolated (SPNT).

Nucleotide Sequencing and Sequence Analysis

Both strands of SPNT cDNA insert were sequenced by the dideoxy chain termination method using the Sequenase DNA sequencing kit and protocol (U. S. Biochemical Corp.). BLAST was used for data base search, and the Genetics Computer Group sequence analysis software package was used to analyze nucleotides and the deduced amino acid sequences.

Northern Blot Analysis

Total RNA from rat liver and other tissues was separated on a 1% formaldehyde-agarose gel and blotted onto a GeneScreen Plus membrane (DuPont NEN). The HindIII-SalI (1958 base pairs) fragment of SPNT that represented coding sequences for SPNT amino acid residues 1-562 was labeled with [P]dCTP (DuPont NEN) using Klenow enzyme (Boehringer Mannheim). Hybridizations were performed at 42 °C in 6 SSPE, 50% formamide, 10 Denhardt's solution, 1% SDS, 10% dextran sulfate, 100 µg/ml salmon sperm DNA. The membranes were washed twice in 2% SSC, 0.5% SDS at room temperature, twice at 1% SSC, 0.5% SDS at 45 °C, and once at 0.2% SSC, 0.2% SDS at 65 °C.


RESULTS AND DISCUSSION

Poly(A) RNA (mRNA) isolated from rat liver gives rise to Na-dependent adenosine uptake when injected into Xenopus oocytes (Fig. 1A). Significant expression of adenosine uptake was observed on day 1 and increased progressively from day 1 to day 3 after mRNA injection. Compared with endogenous transport activity (HO injected or uninjected), 13- and 30-fold increases in Na-dependent adenosine uptake were observed on day 1 and day 3, respectively, after mRNA injection. Augmented Na-dependent adenosine uptake was mainly observed in oocytes injected with larger sizes of mRNA (fractions 1-11) (Fig. 1B). Fractions 5-9 (2-4 kb) were pooled and used to construct a directional cDNA library. To screen the cDNA library, we prepared sense cRNA by in vitro transcription from pools of 1000 clones, injected the cRNA into oocytes, and assayed expression of Na-dependent adenosine uptake. One positive pool was progressively subdivided until a single clone with a cDNA insert of 2.9 kb was isolated. Na-dependent adenosine uptake in cRNA-injected oocytes increased nearly linearly within 120 min (Fig. 2A). At cRNA doses above 1 ng, the expressed transport activities were not linearly related to the amounts of cRNA injected, suggesting that the translation machinery is saturable, which may also explain why the expression level of the isolated clone is not much larger than that of pooled RNA. Competition studies with different purine and pyrimidine nucleosides showed that adenosine uptake was almost completely inhibited by inosine, guanosine, and uridine and only partially inhibited by thymidine and cytidine. SPNT activity was fully inhibited by formycin B, partially inhibited by a purine analog Ara-A, and not inhibited by dipyridamole or azidothymidine (Fig. 2B). The kinetics of SPNT for adenosine and thymidine were compared by examining the initial rates of uptake over a range of concentrations of adenosine and thymidine. Uptake of both nucleosides by the oocytes expressing SPNT was saturable. SPNT has a K of 6 µM for adenosine and 13 µM for thymidine; the V is 457 fmol/oocyte/min for adenosine and 13 fmol/oocyte/min for thymidine. These data indicate that SPNT is the functional Na-dependent purine nucleoside transporter (Cif type) previously characterized in rat liver canalicular plasma membrane (2) .


Figure 1: A, time course of expression of adenosine uptake in X. laevis oocytes. Oocytes were either not injected (, ) or injected with 50 ng of rat liver poly(A) RNA (mRNA) (, ). Uptake of 15 µM [H]adenosine was measured at room temperature (20 °C) in the presence of 100 mM NaCl (, ) or 100 mM choline chloride (, ). B, [H]adenosine uptake in oocytes after 3 days of injection with 50 nl of size-fractionated poly(A) RNA, in the presence of NaCl () or choline chloride (). T, total RNA (50 ng/50 nl); M, mRNA (50 ng/50 nl); 1-24, pools of size-fractionated poly(A) RNA (10-20 ng/50 nl); -, uninjected oocytes. Uptake values are means ± S.E. obtained with 8-10 oocytes. If S.E. bars are not visible they are smaller than the symbols.




Figure 2: A, time course of [H]adenosine uptake in cRNA-injected oocytes. Oocytes were not injected (, ) or injected with 10 ng of synthetic RNA (cRNA) (, ) transcribed from the SPNT clone. Three days after injection, 15 µM [H]adenosine uptake was determined at room temperature (20 °C) over 120 min in the presence of 100 mM NaCl (, ) or choline chloride (, ). B, effects of nucleoside transport inhibitors and nucleoside analogues on adenosine uptake in cRNA-injected oocytes. Oocytes were treated as described in A. 15 µM [H]adenosine uptake was determined at 20 °C in the presence and in the absence (control) of the indicated 1 mM various compounds (, NaCl; , choline chloride). C and D, initial rates of [H]adenosine (C) and [H]thymidine (D) uptake was measured over 1 min using a range of concentrations of 1-1500 µM for adenosine and 1-300 µM for thymidine. Na-specific uptake was determined by subtracting the value in the presence of choline chloride () from the value in the presence of NaCl (). K and V were determined from a double-reciprocal plot (not shown) of these data. Uptake values are mean ± S.E. obtained with 8-10 oocytes. DPA, dipyridamole; AZT, azidothymidine.



We examined the tissue distribution of SPNT expression by Northern blot analysis (Fig. 3). A 2.9-kb transcript was detected in liver, jejunum, spleen, and heart. Additionally, two minor bands corresponding to RNAs of sizes 6 and 1 kb were seen in spleen, jejunum, heart, brain, and skeletal muscle. The function of the latter two transcripts is unknown. The multiple transcripts and tissue expression may relate to diverse function of purine nucleosides, especially adenosine, in various tissues. The transporter may be involved in regulation of purinergic receptor-mediated nucleoside effects in addition to salvaging nucleosides. In contrast, cNT1 was only detected in intestine and kidney, where it may be mainly involved in nucleoside absorption and reabsorption in these two tissues (16) .


Figure 3: Northern blot analysis of SPNT transcript. Total RNAs (25 µg/lane) from kidney (K), spleen (S), jejunum (J), heart (H), brain (B), muscle (M), lung (Lu), and liver (L) tissues were separated by agarose gel electrophoresis and hybridized with the P-labeled SPNT probe as described under ``Experimental Procedures.'' Equal loading was checked by rehybridization with the rRNA probe.



The 2907-base pair sequence of SPNT cDNA contains a single long open reading frame of 1980 nucleotides encoding a protein of 659 amino acid residues with an estimated relative molecular mass of 72 kDa (Fig. 4A). Hydropathy analysis (Fig. 4B) revealed 14 putative membrane-spanning segments. There are five possible N-glycosylation sites (Asn-439, -539, -603, -606, and -625), one ATP/GTP binding motif (GXXXXGKT) in the N terminus, and several consensus sites for protein kinases A (RXS) and C (SXR, SXK, and TXR) phosphorylation in both termini (Fig. 4A). These data suggest that SPNT may be regulated by intracellular ATP/GTP and protein kinases A and C.


Figure 4: A, predicted amino acid sequences of the cDNA, SPNT, encoding the Na-dependent purine nucleoside transporter. Consensus sites for protein kinase A (#) and C (*) phosphorylation and five potential N-linked glycosylation sites (underscored) are shown. B, hydropathy plot using the Kyte-Doolittle method (22) with a window size of 8 amino acids showing putative transmembrane segments 1-14. C, alignment of relevant regions of amino acid sequences of several proteins that share significant homology with SPNT. The locations of these amino acid residues in their respective proteins are indicated by numbers flanking the displayed sequence. Proposed consensus sequences and gaps are displayed in the bottomline, suggesting protein sequence conservation. Sequence data are shown for SPNT, cNT1, ECOHU47_55 yeiM (Escherichia coli) hypothetical 43.4-kDa protein, ECOHU47_52 yeiJ (E. coli) hypothetical 43.4-kDa protein, BSDNPOP_4 (Bacillussubtilis) pyrimidine nucleoside transport protein, ECNUPC_1 NupC (E. coli), and NUPC_ECOLI nucleoside permease NUPC.



An important feature of SPNT is structural similarity (64% identity at the amino acid level) to cNT1 and differences in substrate specificities and tissue distribution. The most divergent regions are in the N- and C-terminal domains, which may be important in tissue-specific regulation. For example, the ATP/GTP binding motif is present in the N terminus of SPNT but not in cNT1. The putative protein kinase A and C phosphorylation sites are also different in locations and numbers. Although the transmembrane regions have similar sequences over most of their length, a number of short stretches of sequence with different amino acids are found. These differences may be sufficient to confer substrate specificity of subtypes of nucleoside transporter as reported for adenosine receptors (21) . The structural similarity of these two proteins suggests that they are members of the same gene family. In contrast, there is no significant homology between SPNT and SNST1.

Comparison with protein sequences in the data bases revealed that several regions of SPNT share significant identity with a number of bacterial membrane proteins, including three nucleoside transport proteins (BSDNPOP_4, ECNUPC_1, and NUPC_ECOLI) and two hypothetical 43.4-kDa proteins (ECOHU47_52 and ECOHU47_55) of unknown function (Fig. 4C). Four consensus sequences (EGSXFVFG, VXSILYYLGLM, NEFVAY, and SXXLXXFANFSSIGIXXG) were derived, which are conserved in sequence and location, i.e. similar space between these consensus sequences. Homology in these regions may reflect common functions, such as facilitating cation or nucleoside binding. We speculate that genes encoding mammalian and bacterial nucleoside transporters may have evolved from the same evolutionary precursors and subsequently underwent extensive evolutionary divergence. The conserved regions may be critical for nucleoside transport function and could be used in searching for additional members of this family.

In summary, we have isolated a bile canalicular Na purine nucleoside co-transporter by expression cloning in Xenopus oocytes. Transcripts were present in multiple tissues. The structural features found in SPNT, cNT1, and bacterial nucleoside transporters suggest that they are members of a single nucleoside transporter superfamily. Several consensus regions that may be important in transport functions were identified.


FOOTNOTES

*
This work was supported by Grant DK-35652 from the National Institutes of Health (to I. M. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U25055.

The abbreviations used are: SPNT, Na-dependent purine nucleoside transporter; kb, kilobase(s).


REFERENCES
  1. Murray, A. W.(1971) Annu. Rev. Biochem. 40, 811-826 [CrossRef][Medline] [Order article via Infotrieve]
  2. Che, M., Nishda, T., Gatmaitan, Z., and Arias, I. M.(1992) J. Biol. Chem. 267, 9684-9688 [Abstract/Free Full Text]
  3. Lin, S. H.(1989) J. Biol. Chem. 264, 14403-14407 [Abstract/Free Full Text]
  4. Charest, R., Blackmore, P. F., and Exton, J. H.(1985) J. Biol. Chem. 260, 15789-15794 [Abstract/Free Full Text]
  5. Shima, S., and Akamatu, N.(1990) Jpn. J. Pharmacol. 53, 473-478 [Medline] [Order article via Infotrieve]
  6. Berhfeld, G., and Forrester, T.(1989) J. Physiol. (Lond.) 418, 88
  7. Forrester, T., and Williams, C. A.(1990) J. Physiol.(Lond.) 268, 371-390 [Medline] [Order article via Infotrieve]
  8. Kitamura, T., Brauneis, U., Gatmaitan, Z., and Arias, I. M.(1991) Hepatology 14, 640-647 [CrossRef][Medline] [Order article via Infotrieve]
  9. Doin, R.(1985) Science 227, 1296-1303 [Medline] [Order article via Infotrieve]
  10. Daval, J., Nehlig, A., and Nicolas, F.(1991) Life Sci. 49, 1435-1453 [CrossRef][Medline] [Order article via Infotrieve]
  11. Plagemann, P. G. W., and Aran, J. M.(1990) Biochim. Biophys. Acta 1028, 289-298 [Medline] [Order article via Infotrieve]
  12. Roden, M., Paterson, A. R. P., and Turnheim, K.(1991) Gastroenterology 100, 1553-1562 [Medline] [Order article via Infotrieve]
  13. Williams, T. C., and Jarvis, S. M.(1991) Biochem. J. 274, 27-33 [Medline] [Order article via Infotrieve]
  14. Vijaualakshmi, D., and Belt, J. A.(1988) J. Biol. Chem. 263, 19419-19423 [Abstract/Free Full Text]
  15. Wu, X., Yuan, G., Brett, C. M., Hui, A. C., and Giacomini, K. M.(1992) J. Biol. Chem. 267, 8813-8818 [Abstract/Free Full Text]
  16. Huang, Q., Yao, S. Y. M., Ritzel, M. W. L., Paterson, A. R. P., Cass, C. E., and Young, J. D.(1994) J. Biol. Chem. 269, 17757-17760 [Abstract/Free Full Text]
  17. Pajor, A. M., and Wright, E. M.(1992) J. Biol. Chem. 267, 3557-3560 [Abstract/Free Full Text]
  18. Chomszynski, P., and Sacchi, N.(1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  19. Palacin, M., Werner, A., Biber, J., and Murer, H.(1990) J. Biol. Chem. 265, 7142-7144 [Abstract/Free Full Text]
  20. Hediger, M. A., Ikeda, T., Coady, M., Gundersen, C. B., and Wright, E. M.(1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2634-2637 [Abstract]
  21. Linden, J.(1994) J. Biol. Chem. 269, 27900-27906 [Abstract/Free Full Text]
  22. Kyte, J., and Doolittle, R.(1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.