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Cloning and Functional Characterization of a Sodium-Dependent Phosphate Transporter Expressed in Human Lung and Small Intestine
Biochemical and Biophysical Research Communications 258, 578 –582 (1999) Article ID bbrc.1999.0666, available online at http://www.idealibrary.com on
Cloning and Functional Characterization of a SodiumDependent Phosphate Transporter Expressed in Human Lung and Small Intestine John A. Feild,* ,1 Li Zhang,* ,2 Kimberly A. Brun,* ,3 David P. Brooks,† and Richard M. Edwards† *Department of Molecular Biology and †Department of Renal Pharmacology, Smithkline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406
Received March 24, 1999
A cDNA clone with 53% amino acid identity to the human type II sodium-dependent phosphate transporter (NaPi-3) was isolated from human small intestine and lung. Functional characterization in Xenopus laevis oocytes showed this cDNA to encode a sodiumdependent phosphate transporter. The electrogenic response is similar to that found in other type II transporters but an inverse pH dependence was observed. By Northern blot, a 4.2-kb transcript was found to be abundantly expressed in lung and, to a lesser degree, in several other tissues of epithelial origin including small intestine, pancreas, prostate, and kidney. This transcript encompasses a 2.073-kb open reading frame which is most closely related (78% amino acid identity) to the mouse sodium-dependent phosphate transporter IIb isoform. This novel transporter, designated human NaPi-3b (Genbank AF111856), appears to be an isoform of the mammalian renal type II co-transporter family. © 1999 Academic Press
Inorganic phosphate absorption from the small intestine and reabsorption from the renal tubule play key roles in the control of inorganic phosphate metabolism and the maintenance of phosphate homeostasis. In the past few years cDNAs encoding sodium-dependent phosphate transporters have been cloned from a number of species (1). Although functionally related, these phosphate transporters are structurally distinct and can be broadly grouped into three sub-types based on sequence identity. Type I transporters represented by 1
To whom correspondence should be addressed at Smithkline Beecham Pharmaceuticals, Dept. of Molecular Biology, UE0548, 709 Swedeland Rd., King of Prussia, PA 19406. Fax: (610) 270-5093. E-mail: [email protected]. 2 Current address: Rhone Poulenc Rorer, 500 Arcola Rd., Collegeville, PA 19426. 3 Current address: Laboratory of Molecular and Cellular Neurobiology, National Institues of Health, NIAAA, Bethesda, MD 20892. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
rabbit NaPi-1 (2) and human and mouse NPT1 (3,4) are found mainly in kidney and do not seem to be regulated by dietary phosphate levels. Several members of the type II transporters have been cloned (4 –7) and show very restricted tissue distribution being found almost exclusively in the renal proximal tubule. One notable exception is the detection of NaPi-3 in osteoclasts (8). A third class of transporter (type III) can also function as a membrane receptor for primate retroviruses (9). Cloning of the type II transporters from renal tubules has significantly advanced our knowledge of molecular mechanisms associated with phosphate reabsorption in the kidney. Although phosphate absorption in the brush border of the small intestine has been documented for some time (10), the exact nature of the transporter(s) responsible has been elusive. Northern blot analysis using type I and type II cDNAs as probes failed to yield any evidence that these transporters are expressed in the small intestine suggesting that the gut may have some as yet unidentified sodiumdependent phosphate transporter system. Searching of an expressed sequence tag (EST) database (11) yielded one EST that was highly homologus to the human sodium-dependent transporter NaPi-3. A full-length clone was obtained from both human small intestine and lung cDNA. Functional characterization of this transporter and analysis of its sequence homology show it to be closely related to NaPi-3 and thus we have named this gene NaPi-3b to indicate that it is an isoform of human NaPi-3. Sequence homology also suggests that human NaPi-3b could be the ortholog of recently described mouse sodium-dependent phosphate transporter IIb (13). MATERIALS AND METHODS Cloning of a novel transporter. A search of an EST database using NaPi-3 (L13258) as a query sequence returned a 549 base pair EST from a small intestinal library (11) with approximately 73%
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amino acid identity to the human sodium-dependent phosphate transporter NaPi-3. Based on the size of the NaPi-3 cDNA sequence, the EST sequence was missing approximately 1000 nucleotides at the 59 end and 300 nucleotides at the 39 end. The 549 base pair cDNA was used as a probe to screen 1 3 10 6 plaques from a lgt10 small intestine cDNA library (Clontech Laboratories, Palo Alto, CA #1133a). A single plaque was isolated after three rounds of screening. The 1.6 kb cDNA insert was subcloned into the pBluescript KS 1 vector (Stratagene, LaJolla, CA) and sequenced. Marathon RACE PCR (Clontech Laboratories, Palo Alto, CA) from small intestine cDNA was used to extend the 39 end of the partial 1.6 kb clone to total length of 2.4 kb. This cDNA sequence encompasses a 2073 nucleotide open reading frame. Using a unique Kpn I site within the cDNA a full-length clone was constructed by consolidation of the overlapping 59 and 39 cDNA sequences. Isolation of full-length cDNA from human lung. Poly A 1 cDNA from human lung (Clontech Laboratories, Palo Alto, CA) was reverse transcribed using the Superscript Pre-amplification system (Life Technologies, Gaithersburg, MD). Oligonucleotide primers were designed based on sequence data obtained from the full-length clone derived from small intestine. Primers just outside the predicted coding region were used in a polymerase chain reaction to amplify a 2.1 kb cDNA. This DNA was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and the insert sequenced in both directions. Northern blot analysis. Multiple tissue blots were purchased from Clontech Laboratories. These blots were probed with 32P labeled cDNA using Clontech ExpressHyb solution, washed in 0.23 SSC (0.03 M NaCl, 0.003 M sodium citrate pH 8.0) twice for 20 minutes each at 65°C and exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY) at 270°C for 16 to 72 hours. Preparation and cRNA microinjection of Xenopus oocytes. Complementary RNA was synthesized in vitro from linearized template cDNA with a MaxiScript RNA transcription kit from Ambion Inc. (Austin, TX). Mature Xenopus laevis frogs were anesthetized by submersion in 0.2% 3-aminobenzoic acid ethyl ester (Sigma Chemical, St. Louis, MO) and a group of oocytes was surgically excised. The oocytes were separated and the follicular cell layer was removed by treatment with type I collagenase (Boehringer Mannheim, Indianapolis, IN) for 2 hours at room temperature. Each oocyte was injected with a total of 50 ng of RNA in 50 nl of diethylpyrocarbonate-treated water. Microinjected oocytes were incubated at 19°C in ND96 solution (96 mM NaCl, 1 mM KCl, 2.0 mM CaCl 2, 0.8 mM Mg Cl 2, 10 mM HEPES, pH 7.4). Oocyte transport assays. Oocytes microinjected with cRNA were analyzed for phosphate transport (3). After 2– 4 days approximately 25 oocytes were washed in sodium free uptake solution (10 mM HEPES, pH 6.6, 100 mM choline chloride, 2 mM KCl, 1 mM CaCl 2, 1 mM MgCl 2) and divided into 2 groups. One was used for analysis of sodium-dependent uptake (using 100 mM NaCl in place of choline chloride) and the other for sodium-independent uptake using the sodium free solution. Oocytes were washed and incubated for 10 to 60 minutes at room temperature in the appropriate buffer containing 0.5 mM KH 2PO 4 (25 mCi/ml 32P-orthophosphoric acid). Following incubation, oocytes were washed 3 times in ice cold sodium free uptake solution containing 5 mM KH 2PO 4, transferred individually to scintillation vials, lysed in 0.2 ml of 10% SDS and counted. Electrophysiological recordings. Two to five days post cRNA microinjection, the oocytes were placed in a 90 ml chamber at room temperature and superfused with ND96 at a rate of approximately 8ml/min. Sodium in the perfusion solution was replaced with choline and applied to the oocytes for a specified time using a solenoid valve. Membrane currents were studied under two-electrode voltage-clamp conditions at a holding potential of 270 mV using an Oocyte Clamp (Warner Instruments, CT). The recording microelectrodes were filled with 3M KCl and had resistances of 0.5–2.0 MV. Data were routinely recorded on a chart recorder.
FIG. 1. Dendrogram of representative members of the type II sodium-dependent phosphate transporter family showing the segregation of the type II transporter family into two distinct groups based on sequence homology. Distances between lines represent evolutionary divergence. Genbank accession numbers used in the alignment are human NaPi-3b: AF111856, mouse type IIb:AF081499 (13), bovine type II:X81699 (5), flounder type II:U13963 (6), rat NaPi-2: L13257 (4), human NaPi-3:L13258 (4), opossum NaPi-4:L26308 (17), mouse NaPi-7:U22465 (18), human NPT1:X71355 (19), human GLVR1:L20859 (20), and rabbit NPT1:M76466 (2). The alignment and dendrogram were created using the Jotun Hein method (21).
RESULTS AND DISCUSSION Isolation and characterization of a full-length cDNA. An EST was found in a small intestine cDNA library which aligned with human NaPi-3. The region of identity overlapped amino acids 351 to 534 of NaPi-3 which encodes transmembrane regions 4 through 8 of the predicted 8 transmembrane domains of NaPi-3. This alignment indicated that an isoform of NaPi-3 might exist. Library screening and RACE PCR resulted in the isolation of a cDNA encoding a 2073 base pair open reading frame. A BLAST (12) sequence alignment indicated this 2.073 kb clone had higher homology to the bovine NaPi transporter (5) than to the human NaPi-3. Sequence alignment of the full-length clone with other type II transporters showed that the amino acid identity ranged between 50% and 78%. Highest identity (78%) was found to the recently described mouse type IIb isoform (13) and to the bovine (76%) and flounder (61%) type II co-transporters. Multiple sequence alignments with other members of the type II family indicates that this family segregates into two groups as shown in Fig. 1. One group, exemplified by NaPi-2 and NaPi-3 are found almost exclusively in the renal proximal tubules while the subject of this report, human NaPi-3b, aligns with a second group of more widely distributed transporters. The highest degree of homology amongst all members of the type II family is found in the region of the transmembrane domains while there is significant divergence at the amino and carboxy termini. In addition, NaPi-3b is cysteine rich in the amino terminal portion (Fig. 2), similar to bo-
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FIG. 2. Multiple sequence alignment of human NaPi-3b, bovine type II, and human NaPi-3. Residues conserved in all three proteins are shaded. Residues shared with NaPi3b are boxed. Potential glycosylation sites in extracellular loops are marked with an asterisk. A line overscores transmembrane spanning domains.
vine, flounder and mouse IIb while rat NaPi-2 and human NaPi-3 are lacking this cysteine rich region. This analysis suggests NaPi-3b to be an isoform of type II transporters and an ortholog of mouse type IIb. Northern blot analysis and RT-PCR. Probing of multiple tissue blots using NaPi-3b cDNA as probe shows an approximately 4.2 kilobase mRNA expressed in many cell types of epithelial origin (Fig. 3). Although the transcript size is 4.2 kb the open reading frame of 2073 nucleotides suggests a 59 untranslated region of almost 2 kilobases. This transcript size for human NaPi-3b is similar to a transcript of about 4 kb as reported for mouse IIb which has a coding region of 2.1 kb. Slight size differences of the transcript are seen between different tissues. Expression is very high in the lung, with moderate levels seen in the prostate, pancreas and kidney and lower levels in small intestine, ovary, testis, and prostate. No signal was detected in mRNA from heart, brain, spleen, peripheral blood lymphocytes, colon, or skeletal muscle after prolonged exposure. The actual degree of expression in any particular cell type may be under-represented on these blots since the samples are total tissue extracts and not
fractionated (e.g. serosal and mucosal) cell populations. In situ hybridization experiments can better address the issue of the cellular localization of NaPi-3b.
FIG. 3. Northern blot analysis of NaPi-3b expression. Multiple tissue blots (approximately 2 mg Poly(A) 1 per lane) were hybridized with a 32P labeled cDNA probe and processed as described under Materials and Methods. Autoradiographs were exposed for 48 h at 270°C (panels A and C) or 16 h (panel B). Panel A: spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood lymphocytes. Panel B: heart, brain, lung, and liver. Panel C: smooth muscle, kidney, and pancreas.
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may play a role in phosphate and phospholipid metabolism during the production of pulmonary surfactants. In order to confirm that is NaPi-3b in lung and not a related cross hybridizing transcript the polymerase chain reaction was used to amplify a DNA fragment from lung mRNA. A 2.1 kilobase cDNA was subcloned and sequencing confirmed the expression of NaPi-3b in lung tissue. Kidney mRNA shows a strongly hybridizing second band at approximately 2.4 kb which may be a processed form of the transcript. Further studies will be needed to determine the exact nature of this lower molecular weight kidney mRNA transcript.
FIG. 4. Phosphate uptake in microinjected Xenopus oocytes. Three days postinjection of 25 ng cRNA the oocytes was assayed for phosphate uptake as described under Materials and Methods. Uptake in the presence of sodium (100 mM) is indicated by shaded bars, and uptake in sodium free buffer (100 mM choline chloride) is indicated by open bars. Each bar represents the average of 12 oocytes.
The high level of expression of NaPi-3b in the lung has not been seen with other transporters. A transcript of about 5 kb was reported (4) in lung when using NaPi-3 as probe and it is possible that this 5 kb lung transcript is actually NaPi-3b detected by cross hybridization of the NaPi-3 probe. A sodium-dependent phosphate transporter system has been functionally detected in rat lung alveolar epithelial cells (14) which
Oocyte transport assays and electrophysiology. To evaluate the function of the novel transporter Xenopus oocytes were microinjected with NaPi-3b cRNA. Two to three days post-injection an approximately 10-fold increase of phosphate uptake was seen in NaPi-3b oocytes compared to oocytes injected with water (Fig. 4). The presence of sodium is an absolute requirement for the increase of phosphate uptake. This data is similar for that reported for other sodium-dependent phosphate transporters (3,4) and clearly demonstrates that NaPi-3b is a sodium-dependent phosphate transporter. To determine the function of NaPi-3b on a single cell basis, we applied a two-electrode voltage clamp technique to record the currents following activation of NaPi-3b. When oocytes expressing NaPi-3b were superfused with sodium phosphate inward currents of 15 to 100 nA were recorded comparable to the response seen for other type II transporters (16,18). Similar to
FIG. 5. Electrophysiological measurement of pH dependence of NaPi-3b. Oocytes were prepared as noted in Fig. 4. The trace records were recorded at 270 mV in the same oocyte and represent the inward currents upon superfusion in the presence of sodium at different pH levels. The bars on top of each record indicate the time of the application of the sodium containing solution. 581
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results seen using isotope in oocyte phosphate transport assays the electrogenic response of the oocytes is sodium-dependent (data not shown). Interestingly, the uptake of phosphate by NaPi-3b exhibits a strong sensitivity to pH as shown in Fig. 5. Phosphate uptake was increased at more acidic pH levels, being significantly higher at pH 6.6 than 7.0, whereas pH levels above 7.0 showed phosphate uptake to be barely above background levels. This acidic pH dependence has been reported using rabbit small intestine brush border membrane vesicle assays (14) and is consistent with results found in mouse type IIb (13). In both these studies, at saturating levels of sodium, the rate of phosphate uptake was found to be higher at more acidic pH levels. However, the opposite pH dependence is found to be associated with type II transporters expressed in kidney (15,16). The inverse pH dependence is also seen with mouse type IIb. The type II transporters can be segregated into two groups based not only on sequence homology but also tissue distribution. In addition, the inverse pH relationship of the mouse type IIb and human NaPi-3b underscores the phylogenetic differences between these two sub-groups. This report has described the cloning of a novel human isoform of a sodium-dependent phosphate transporter. It was found to possess the characteristics previously documented for Na/P i co-transport in small intestine brush border membranes. Further studies using in situ hybridization and immunohistochemistry should provide more insight into the nature of this co-transporter. Although NaPi-3b may be the small intestine Na/P i co-transporter its wide tissue distribution suggests that could play a role in phosphate metabolism of many cell types. REFERENCES 1. Murer, H., and Biber, J. (1996) Annu. Rev. Physiol. 58, 607– 618. 2. Werner, A., Moore, M., Mantei, N., Biber, J., Semenza, G., and Murer, H. (1991) Proc. Natl. Acad. Sci. USA 88, 9608 –9612.
3. Miyamoto, K., Tatsumi, S., Sonoda, T., Yamamoto, H., Minami, H., and Taketani, Y. (1995) Biochem. J. 305, 81– 85. 4. Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stage, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. USA 90, 5979 –5983. 5. Helps, C., Murer, H., and McGivan, J. (1995) Eur. J. Biochem. 228, 927–930. 6. Werner, A., Murer, H., and Kinne, R. (1994) Am. J. Physiol. F311–F317. 7. Verri, T., Markovich, D., Perego, C., Norbis, F., Stange, G., Sorribas, V., Biber, J., and Murer, H. (1995) Am. J. Physiol. 268, F626 –F633. 8. Gupta, A., Guo, X. L., Alvarez, U. M., and Hruska, K. A. (1997) J. Clin. Invest. 100, 539 –549. 9. Olah, Z., Lehel, C., Anderson, W. B., Eiden, M. V., and Wilson, C. A. (1994) J. Biol. Chem. 269, 25426 –25431. 10. Berner, W., Kinne, R., and Murer, H. (1976) Biochem. J. 160, 467– 474. 11. Adams, M. D., Dubnick, Kerlavage, A. R., Moreno, R., Kelley, J. M., Kelly, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992) Nature 355, 632– 634. 12. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403– 410. 13. Hilfiker, H., Hattenhauer, O., Traebert, M., Forster, I., Murer, H., and Biber, J. (1998) Proc. Natl. Acad. Sci. USA 95, 14564 –14569. 14. Clerici, C., Soler, P., and Saumon, G. (1991) Biochemica et Biophysica Acta 1063, 27–35. 15. Danisi, G., Murer, H., Straub, R. W. (1984) Am. J. Physiol. 246, G180 –186. 16. Busch, A., Waldegger, S., Herzer, T., Biber, J., Markovich, D., Hayes, G., Murer, H., and Lang, F. (1994) Proc. Natl. Acad. Sci. USA 91, 8205– 8208. 17. Sorribas, V., Markovich, D., Hayes, G., Stange, G., Forgo, J., Biber, J., and Murer, H. (1994) J. Biol. Chem. 269, 6615– 6621. 18. Hartmann, C. M., Wagner, C. A., Busch, A. E., Markovich, D., Biber, J., Lang, F., and Murer, H. (1995) Pflugers Arch 430, 830 – 836. 19. Chong, S. S., Kristjansson, K., Zoghbi, H. Y., and Hughes, M. R. (1993) Genomics 18, 355–359. 20. O’Hara, B., Johann, S. V., Klinger, H. P., Blair, D. G., Rubinson, H., Dunn, K. J., Sass, P., Vitek, S. M., and Robins, T. (1990) Cell Growth Differ. 1, 119 –127. 21. Hein, J. (1990) Meth. Enzymology 183, 626 – 645.
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Cloning and Functional Characterization of a SodiumDependent Phosphate Transporter Expressed in Human Lung and Small Intestine John A. Feild,* ,1 Li Zhang,* ,2 Kimberly A. Brun,* ,3 David P. Brooks,† and Richard M. Edwards† *Department of Molecular Biology and †Department of Renal Pharmacology, Smithkline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406
Received March 24, 1999
A cDNA clone with 53% amino acid identity to the human type II sodium-dependent phosphate transporter (NaPi-3) was isolated from human small intestine and lung. Functional characterization in Xenopus laevis oocytes showed this cDNA to encode a sodiumdependent phosphate transporter. The electrogenic response is similar to that found in other type II transporters but an inverse pH dependence was observed. By Northern blot, a 4.2-kb transcript was found to be abundantly expressed in lung and, to a lesser degree, in several other tissues of epithelial origin including small intestine, pancreas, prostate, and kidney. This transcript encompasses a 2.073-kb open reading frame which is most closely related (78% amino acid identity) to the mouse sodium-dependent phosphate transporter IIb isoform. This novel transporter, designated human NaPi-3b (Genbank AF111856), appears to be an isoform of the mammalian renal type II co-transporter family. © 1999 Academic Press
Inorganic phosphate absorption from the small intestine and reabsorption from the renal tubule play key roles in the control of inorganic phosphate metabolism and the maintenance of phosphate homeostasis. In the past few years cDNAs encoding sodium-dependent phosphate transporters have been cloned from a number of species (1). Although functionally related, these phosphate transporters are structurally distinct and can be broadly grouped into three sub-types based on sequence identity. Type I transporters represented by 1
To whom correspondence should be addressed at Smithkline Beecham Pharmaceuticals, Dept. of Molecular Biology, UE0548, 709 Swedeland Rd., King of Prussia, PA 19406. Fax: (610) 270-5093. E-mail: [email protected]. 2 Current address: Rhone Poulenc Rorer, 500 Arcola Rd., Collegeville, PA 19426. 3 Current address: Laboratory of Molecular and Cellular Neurobiology, National Institues of Health, NIAAA, Bethesda, MD 20892. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
rabbit NaPi-1 (2) and human and mouse NPT1 (3,4) are found mainly in kidney and do not seem to be regulated by dietary phosphate levels. Several members of the type II transporters have been cloned (4 –7) and show very restricted tissue distribution being found almost exclusively in the renal proximal tubule. One notable exception is the detection of NaPi-3 in osteoclasts (8). A third class of transporter (type III) can also function as a membrane receptor for primate retroviruses (9). Cloning of the type II transporters from renal tubules has significantly advanced our knowledge of molecular mechanisms associated with phosphate reabsorption in the kidney. Although phosphate absorption in the brush border of the small intestine has been documented for some time (10), the exact nature of the transporter(s) responsible has been elusive. Northern blot analysis using type I and type II cDNAs as probes failed to yield any evidence that these transporters are expressed in the small intestine suggesting that the gut may have some as yet unidentified sodiumdependent phosphate transporter system. Searching of an expressed sequence tag (EST) database (11) yielded one EST that was highly homologus to the human sodium-dependent transporter NaPi-3. A full-length clone was obtained from both human small intestine and lung cDNA. Functional characterization of this transporter and analysis of its sequence homology show it to be closely related to NaPi-3 and thus we have named this gene NaPi-3b to indicate that it is an isoform of human NaPi-3. Sequence homology also suggests that human NaPi-3b could be the ortholog of recently described mouse sodium-dependent phosphate transporter IIb (13). MATERIALS AND METHODS Cloning of a novel transporter. A search of an EST database using NaPi-3 (L13258) as a query sequence returned a 549 base pair EST from a small intestinal library (11) with approximately 73%
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amino acid identity to the human sodium-dependent phosphate transporter NaPi-3. Based on the size of the NaPi-3 cDNA sequence, the EST sequence was missing approximately 1000 nucleotides at the 59 end and 300 nucleotides at the 39 end. The 549 base pair cDNA was used as a probe to screen 1 3 10 6 plaques from a lgt10 small intestine cDNA library (Clontech Laboratories, Palo Alto, CA #1133a). A single plaque was isolated after three rounds of screening. The 1.6 kb cDNA insert was subcloned into the pBluescript KS 1 vector (Stratagene, LaJolla, CA) and sequenced. Marathon RACE PCR (Clontech Laboratories, Palo Alto, CA) from small intestine cDNA was used to extend the 39 end of the partial 1.6 kb clone to total length of 2.4 kb. This cDNA sequence encompasses a 2073 nucleotide open reading frame. Using a unique Kpn I site within the cDNA a full-length clone was constructed by consolidation of the overlapping 59 and 39 cDNA sequences. Isolation of full-length cDNA from human lung. Poly A 1 cDNA from human lung (Clontech Laboratories, Palo Alto, CA) was reverse transcribed using the Superscript Pre-amplification system (Life Technologies, Gaithersburg, MD). Oligonucleotide primers were designed based on sequence data obtained from the full-length clone derived from small intestine. Primers just outside the predicted coding region were used in a polymerase chain reaction to amplify a 2.1 kb cDNA. This DNA was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and the insert sequenced in both directions. Northern blot analysis. Multiple tissue blots were purchased from Clontech Laboratories. These blots were probed with 32P labeled cDNA using Clontech ExpressHyb solution, washed in 0.23 SSC (0.03 M NaCl, 0.003 M sodium citrate pH 8.0) twice for 20 minutes each at 65°C and exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY) at 270°C for 16 to 72 hours. Preparation and cRNA microinjection of Xenopus oocytes. Complementary RNA was synthesized in vitro from linearized template cDNA with a MaxiScript RNA transcription kit from Ambion Inc. (Austin, TX). Mature Xenopus laevis frogs were anesthetized by submersion in 0.2% 3-aminobenzoic acid ethyl ester (Sigma Chemical, St. Louis, MO) and a group of oocytes was surgically excised. The oocytes were separated and the follicular cell layer was removed by treatment with type I collagenase (Boehringer Mannheim, Indianapolis, IN) for 2 hours at room temperature. Each oocyte was injected with a total of 50 ng of RNA in 50 nl of diethylpyrocarbonate-treated water. Microinjected oocytes were incubated at 19°C in ND96 solution (96 mM NaCl, 1 mM KCl, 2.0 mM CaCl 2, 0.8 mM Mg Cl 2, 10 mM HEPES, pH 7.4). Oocyte transport assays. Oocytes microinjected with cRNA were analyzed for phosphate transport (3). After 2– 4 days approximately 25 oocytes were washed in sodium free uptake solution (10 mM HEPES, pH 6.6, 100 mM choline chloride, 2 mM KCl, 1 mM CaCl 2, 1 mM MgCl 2) and divided into 2 groups. One was used for analysis of sodium-dependent uptake (using 100 mM NaCl in place of choline chloride) and the other for sodium-independent uptake using the sodium free solution. Oocytes were washed and incubated for 10 to 60 minutes at room temperature in the appropriate buffer containing 0.5 mM KH 2PO 4 (25 mCi/ml 32P-orthophosphoric acid). Following incubation, oocytes were washed 3 times in ice cold sodium free uptake solution containing 5 mM KH 2PO 4, transferred individually to scintillation vials, lysed in 0.2 ml of 10% SDS and counted. Electrophysiological recordings. Two to five days post cRNA microinjection, the oocytes were placed in a 90 ml chamber at room temperature and superfused with ND96 at a rate of approximately 8ml/min. Sodium in the perfusion solution was replaced with choline and applied to the oocytes for a specified time using a solenoid valve. Membrane currents were studied under two-electrode voltage-clamp conditions at a holding potential of 270 mV using an Oocyte Clamp (Warner Instruments, CT). The recording microelectrodes were filled with 3M KCl and had resistances of 0.5–2.0 MV. Data were routinely recorded on a chart recorder.
FIG. 1. Dendrogram of representative members of the type II sodium-dependent phosphate transporter family showing the segregation of the type II transporter family into two distinct groups based on sequence homology. Distances between lines represent evolutionary divergence. Genbank accession numbers used in the alignment are human NaPi-3b: AF111856, mouse type IIb:AF081499 (13), bovine type II:X81699 (5), flounder type II:U13963 (6), rat NaPi-2: L13257 (4), human NaPi-3:L13258 (4), opossum NaPi-4:L26308 (17), mouse NaPi-7:U22465 (18), human NPT1:X71355 (19), human GLVR1:L20859 (20), and rabbit NPT1:M76466 (2). The alignment and dendrogram were created using the Jotun Hein method (21).
RESULTS AND DISCUSSION Isolation and characterization of a full-length cDNA. An EST was found in a small intestine cDNA library which aligned with human NaPi-3. The region of identity overlapped amino acids 351 to 534 of NaPi-3 which encodes transmembrane regions 4 through 8 of the predicted 8 transmembrane domains of NaPi-3. This alignment indicated that an isoform of NaPi-3 might exist. Library screening and RACE PCR resulted in the isolation of a cDNA encoding a 2073 base pair open reading frame. A BLAST (12) sequence alignment indicated this 2.073 kb clone had higher homology to the bovine NaPi transporter (5) than to the human NaPi-3. Sequence alignment of the full-length clone with other type II transporters showed that the amino acid identity ranged between 50% and 78%. Highest identity (78%) was found to the recently described mouse type IIb isoform (13) and to the bovine (76%) and flounder (61%) type II co-transporters. Multiple sequence alignments with other members of the type II family indicates that this family segregates into two groups as shown in Fig. 1. One group, exemplified by NaPi-2 and NaPi-3 are found almost exclusively in the renal proximal tubules while the subject of this report, human NaPi-3b, aligns with a second group of more widely distributed transporters. The highest degree of homology amongst all members of the type II family is found in the region of the transmembrane domains while there is significant divergence at the amino and carboxy termini. In addition, NaPi-3b is cysteine rich in the amino terminal portion (Fig. 2), similar to bo-
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FIG. 2. Multiple sequence alignment of human NaPi-3b, bovine type II, and human NaPi-3. Residues conserved in all three proteins are shaded. Residues shared with NaPi3b are boxed. Potential glycosylation sites in extracellular loops are marked with an asterisk. A line overscores transmembrane spanning domains.
vine, flounder and mouse IIb while rat NaPi-2 and human NaPi-3 are lacking this cysteine rich region. This analysis suggests NaPi-3b to be an isoform of type II transporters and an ortholog of mouse type IIb. Northern blot analysis and RT-PCR. Probing of multiple tissue blots using NaPi-3b cDNA as probe shows an approximately 4.2 kilobase mRNA expressed in many cell types of epithelial origin (Fig. 3). Although the transcript size is 4.2 kb the open reading frame of 2073 nucleotides suggests a 59 untranslated region of almost 2 kilobases. This transcript size for human NaPi-3b is similar to a transcript of about 4 kb as reported for mouse IIb which has a coding region of 2.1 kb. Slight size differences of the transcript are seen between different tissues. Expression is very high in the lung, with moderate levels seen in the prostate, pancreas and kidney and lower levels in small intestine, ovary, testis, and prostate. No signal was detected in mRNA from heart, brain, spleen, peripheral blood lymphocytes, colon, or skeletal muscle after prolonged exposure. The actual degree of expression in any particular cell type may be under-represented on these blots since the samples are total tissue extracts and not
fractionated (e.g. serosal and mucosal) cell populations. In situ hybridization experiments can better address the issue of the cellular localization of NaPi-3b.
FIG. 3. Northern blot analysis of NaPi-3b expression. Multiple tissue blots (approximately 2 mg Poly(A) 1 per lane) were hybridized with a 32P labeled cDNA probe and processed as described under Materials and Methods. Autoradiographs were exposed for 48 h at 270°C (panels A and C) or 16 h (panel B). Panel A: spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood lymphocytes. Panel B: heart, brain, lung, and liver. Panel C: smooth muscle, kidney, and pancreas.
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may play a role in phosphate and phospholipid metabolism during the production of pulmonary surfactants. In order to confirm that is NaPi-3b in lung and not a related cross hybridizing transcript the polymerase chain reaction was used to amplify a DNA fragment from lung mRNA. A 2.1 kilobase cDNA was subcloned and sequencing confirmed the expression of NaPi-3b in lung tissue. Kidney mRNA shows a strongly hybridizing second band at approximately 2.4 kb which may be a processed form of the transcript. Further studies will be needed to determine the exact nature of this lower molecular weight kidney mRNA transcript.
FIG. 4. Phosphate uptake in microinjected Xenopus oocytes. Three days postinjection of 25 ng cRNA the oocytes was assayed for phosphate uptake as described under Materials and Methods. Uptake in the presence of sodium (100 mM) is indicated by shaded bars, and uptake in sodium free buffer (100 mM choline chloride) is indicated by open bars. Each bar represents the average of 12 oocytes.
The high level of expression of NaPi-3b in the lung has not been seen with other transporters. A transcript of about 5 kb was reported (4) in lung when using NaPi-3 as probe and it is possible that this 5 kb lung transcript is actually NaPi-3b detected by cross hybridization of the NaPi-3 probe. A sodium-dependent phosphate transporter system has been functionally detected in rat lung alveolar epithelial cells (14) which
Oocyte transport assays and electrophysiology. To evaluate the function of the novel transporter Xenopus oocytes were microinjected with NaPi-3b cRNA. Two to three days post-injection an approximately 10-fold increase of phosphate uptake was seen in NaPi-3b oocytes compared to oocytes injected with water (Fig. 4). The presence of sodium is an absolute requirement for the increase of phosphate uptake. This data is similar for that reported for other sodium-dependent phosphate transporters (3,4) and clearly demonstrates that NaPi-3b is a sodium-dependent phosphate transporter. To determine the function of NaPi-3b on a single cell basis, we applied a two-electrode voltage clamp technique to record the currents following activation of NaPi-3b. When oocytes expressing NaPi-3b were superfused with sodium phosphate inward currents of 15 to 100 nA were recorded comparable to the response seen for other type II transporters (16,18). Similar to
FIG. 5. Electrophysiological measurement of pH dependence of NaPi-3b. Oocytes were prepared as noted in Fig. 4. The trace records were recorded at 270 mV in the same oocyte and represent the inward currents upon superfusion in the presence of sodium at different pH levels. The bars on top of each record indicate the time of the application of the sodium containing solution. 581
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results seen using isotope in oocyte phosphate transport assays the electrogenic response of the oocytes is sodium-dependent (data not shown). Interestingly, the uptake of phosphate by NaPi-3b exhibits a strong sensitivity to pH as shown in Fig. 5. Phosphate uptake was increased at more acidic pH levels, being significantly higher at pH 6.6 than 7.0, whereas pH levels above 7.0 showed phosphate uptake to be barely above background levels. This acidic pH dependence has been reported using rabbit small intestine brush border membrane vesicle assays (14) and is consistent with results found in mouse type IIb (13). In both these studies, at saturating levels of sodium, the rate of phosphate uptake was found to be higher at more acidic pH levels. However, the opposite pH dependence is found to be associated with type II transporters expressed in kidney (15,16). The inverse pH dependence is also seen with mouse type IIb. The type II transporters can be segregated into two groups based not only on sequence homology but also tissue distribution. In addition, the inverse pH relationship of the mouse type IIb and human NaPi-3b underscores the phylogenetic differences between these two sub-groups. This report has described the cloning of a novel human isoform of a sodium-dependent phosphate transporter. It was found to possess the characteristics previously documented for Na/P i co-transport in small intestine brush border membranes. Further studies using in situ hybridization and immunohistochemistry should provide more insight into the nature of this co-transporter. Although NaPi-3b may be the small intestine Na/P i co-transporter its wide tissue distribution suggests that could play a role in phosphate metabolism of many cell types. REFERENCES 1. Murer, H., and Biber, J. (1996) Annu. Rev. Physiol. 58, 607– 618. 2. Werner, A., Moore, M., Mantei, N., Biber, J., Semenza, G., and Murer, H. (1991) Proc. Natl. Acad. Sci. USA 88, 9608 –9612.
3. Miyamoto, K., Tatsumi, S., Sonoda, T., Yamamoto, H., Minami, H., and Taketani, Y. (1995) Biochem. J. 305, 81– 85. 4. Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stage, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. USA 90, 5979 –5983. 5. Helps, C., Murer, H., and McGivan, J. (1995) Eur. J. Biochem. 228, 927–930. 6. Werner, A., Murer, H., and Kinne, R. (1994) Am. J. Physiol. F311–F317. 7. Verri, T., Markovich, D., Perego, C., Norbis, F., Stange, G., Sorribas, V., Biber, J., and Murer, H. (1995) Am. J. Physiol. 268, F626 –F633. 8. Gupta, A., Guo, X. L., Alvarez, U. M., and Hruska, K. A. (1997) J. Clin. Invest. 100, 539 –549. 9. Olah, Z., Lehel, C., Anderson, W. B., Eiden, M. V., and Wilson, C. A. (1994) J. Biol. Chem. 269, 25426 –25431. 10. Berner, W., Kinne, R., and Murer, H. (1976) Biochem. J. 160, 467– 474. 11. Adams, M. D., Dubnick, Kerlavage, A. R., Moreno, R., Kelley, J. M., Kelly, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992) Nature 355, 632– 634. 12. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403– 410. 13. Hilfiker, H., Hattenhauer, O., Traebert, M., Forster, I., Murer, H., and Biber, J. (1998) Proc. Natl. Acad. Sci. USA 95, 14564 –14569. 14. Clerici, C., Soler, P., and Saumon, G. (1991) Biochemica et Biophysica Acta 1063, 27–35. 15. Danisi, G., Murer, H., Straub, R. W. (1984) Am. J. Physiol. 246, G180 –186. 16. Busch, A., Waldegger, S., Herzer, T., Biber, J., Markovich, D., Hayes, G., Murer, H., and Lang, F. (1994) Proc. Natl. Acad. Sci. USA 91, 8205– 8208. 17. Sorribas, V., Markovich, D., Hayes, G., Stange, G., Forgo, J., Biber, J., and Murer, H. (1994) J. Biol. Chem. 269, 6615– 6621. 18. Hartmann, C. M., Wagner, C. A., Busch, A. E., Markovich, D., Biber, J., Lang, F., and Murer, H. (1995) Pflugers Arch 430, 830 – 836. 19. Chong, S. S., Kristjansson, K., Zoghbi, H. Y., and Hughes, M. R. (1993) Genomics 18, 355–359. 20. O’Hara, B., Johann, S. V., Klinger, H. P., Blair, D. G., Rubinson, H., Dunn, K. J., Sass, P., Vitek, S. M., and Robins, T. (1990) Cell Growth Differ. 1, 119 –127. 21. Hein, J. (1990) Meth. Enzymology 183, 626 – 645.
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