cotransporter (lac permease) in Escherichia coli (1) and the. Na+/glucose cotransporter in intestinal brush borders (2). Two prokaryote Na+ cotransporters have ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5748-5752, August 1989 Biochemistry
Homology of the human intestinal Na+/glucose and Escherichia coli Na /proline cotransporters (symport/amino acid sequences/structure-function relations)
MATTHIAS A. HEDIGER, ERIC TURK, AND ERNEST M. WRIGHT Department of Physiology, University of California at Los Angeles School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90024-1751
Communicated by H. Ronald Kaback, May 3, 1989 (received for review March 17, 1989)
vol) formamide, and the filter was washed at high stringency [650C in the presence of 0.1 x SSC (1 x SSC is 0.15 M sodium chloride/0.015 M sodium citrate, pH 7)/0.1% SDS].
ABSTRACT Cotransport proteins are responsible for the active accumulation of organic substrates in cells. Na+ gradients provide the driving force for uptake of most substrates into eukaryotes and for a few substrates in some prokaryotes. We report here the cloning and sequencing of the human intestinal Na+/glucose cotransporter (SGLTI) and compare its structure with other cloned transporters. At the DNA level and the predicted amino acid and secondary structure levels, dose homology is evident between the human and rabbit intestinal Na+/glucose cotransporters, and a sigificant homology is found between these and the Escherichia coli Na+/proline cotransporter (putP). No homology is detectible with other known proteins. We infer from these results that the mammalian Nat/glucose and prokaryote Na+/proline cotransporters share a common ancestral gene.
RESULTS AND DISCUSSION A 2.6-kb cDNA clone for the human intestinal Na+/glucose cotransporter was selected from the cDNA library using the rabbit intestinal Na+/glucose cotransporter cDNA clone (6) as a probe. mRNA synthesized in vitro from the clone and injected into Xenopus oocytes increased the Na'-dependent, phlorizin-sensitive sugar uptake >70-fold above that of control water-injected oocytes. In the presence of sodium, the rate of 50 AM a-methyl-D-[14C]glucopyranoside uptake increased from 0.7 ± 0.1 to 74 ± 19 pmol per oocyte per hr in six experiments. Transport was inhibited >80% by 25 AM phlorizin and by 25 mM D-galactose. The DNA and deduced amino acid sequences of the clone are presented in Fig. 1. There is 94% similarity between the human and rabbit amino acid sequences (6), 84% of the residues being identical and another 10% being conservative substitutions. Among the notable differences are substitutions for lysine at positions 139, 596, 616, and 622 and for tyrosine at positions 20, 246, and 660. This result indicates that these residues are not among the important lysine and tyrosine residues previously identified at the glucose and Na+ binding sites (7, 8). At the DNA level, an 82% similarity is evident between the sequences of the rabbit and human clones. This similarity is reflected in the results of Northern blot analysis of human and rabbit mRNA using the full-length cDNA coding for the human intestinal Na+/glucose cotransporter as a probe (Fig. 2). The probe hybridized with 2.3-kb rabbit intestinal mRNA, which matches the size of mRNA coding for the cotransporter (6). Three bands of human mRNA (2.2, 2.6, and 4.8 kb) hybridized to the probe. We have isolated clones with inserts of these three sizes from the human intestinal library and found all to contain the same coding region with different lengths of the 3' noncoding region. The deduced amino acid sequence was analyzed for potential membrane-spanning regions using hydrophobic moment analysis (Fig. 3). There are 11 predicted membranespanning regions and 2 highly charged hydrophilic regions (residues 408-420 and 567-631). These regions are similar to those predicted for the rabbit intestinal Na+/glucose cotransporter (6). An additional putative membrane segment, 7a, is indicated for the human sequence; it is due to the replacement of Met-387 in rabbit by a less polar leucine. Another hydrophobic segment, 9a, is also shown, but its hydrophobic moment is relatively high (it contains an arginine at residue 499), and it is probably a membrane surface component. There are two potential N-linked glycosylation sites (AsnXaa-Thr/Ser) at positions 248 and 306. We have evidence that only Asn-248 is glycosylated in the rabbit (10), thereby
In both prokaryotes and eukaryotes the active accumulation of organic substrates occurs by cotransport (symport) systems. These systems are driven mainly by electrochemical potential gradients of H+ in enteric bacteria and of Na+ in eukaryotes. The archetypal transporters are the H+/lactose cotransporter (lac permease) in Escherichia coli (1) and the Na+/glucose cotransporter in intestinal brush borders (2). Two prokaryote Na+ cotransporters have been identified, cloned, and sequenced-the E. coli melibiose (meiB) and proline (putP) carriers (3, 4). There is no homology between meiB and putP and between these Na+ cotransporters and H+ cotransporters such as lac permease. We have cloned the human intestinal Na+/glucose cotransporter (SGLTJ)* and find clear homology with the putP, but not the meiB transporter, indicating an evolutionary link between bacterial and human Na+-cotransport proteins.
METHODS A human intestinal cDNA AgtlO library was constructed from ileum mRNA (5), and clones were selected using the rabbit intestinal Na+/glucose cotransporter cDNA (6) as a probe. A positive clone with a 2.6-kilobase (kb) insert was subcloned into Bluescript plasmid for in vitro transcription and into M13mpl8 and -mpl9 vectors for Sanger dideoxynucleotide sequencing. RNA synthesized from the Bluescript plasmid in vitro was injected into Xenopus oocytes, and the expression of the Na+/glucose cotransporter was tested by measuring the Na+-dependent uptake of a-methyl-D-['4C]glucopyranoside. All methods including DNA sequencing by the Sanger method were performed as described (6). For Northern (RNA) analysis, the RNA gel was run in the presence of 2.2 M formaldehyde (2.5 ug of RNA per lane). The gel was capillary blotted to a nitrocellulose filter (Schleicher & Schuell). Hybridization was performed in 50% (vol/ The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*The sequence reported in this paper has been deposited in the GenBank data base (accession no. M24847). 5748
Proc. Nati. Acad. Sci. USA 86 (1989)
Biochemistry: Hediger et al. 10 Rabbit Human
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5750
Biochemistry: Hediger et al.
Proc. Natl. Acad. Sci. USA 86 (1989)
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suggesting that the N terminus of the protein is on the cytoplasmic face of the membrane (Fig. 3). The GenBank database (Release 57.0) was searched for sequences related to the human Na+/glucose cotransporter using the program TFASTA (11). We found a significant similarity to the E. coli Na+/proline cotransporter. The sequence alignment of the Na+/proline cotransporter and the human Na+/glucose cotransporter, obtained using the program BESTFIT (12), is presented in Fig. 4. There is 28% identity between the amino acid sequences of the two transporters, and there are conservative substitutions at another 25% of the residues. The significance of this alignment was evaluated using the program RDF (14) and is nine SD units. Similar values were obtained using the programs SEQDP (15) and ALIGN (16). Comparison of the human Na+/glucose and E. coli melibiose transport proteins yielded a SD of -0.66. Because the SD must be >3-5 to show significant similarity between two sequences (17), we conclude that the Na+/ glucose transporter is homologous to the Na+/proline trans-
9
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porter but not to the Na+/melibiose transporter. At the DNA level, there is to 44% similarity between the Na+/glucose and Na+/proline transporter clones. This degree of similarity is comparable to that noted previously between the mammalian facilitated glucose transporter and the E. coli H+/arabinose and H+/xylose cotransporters (18). The three sequences have -30% amino acid identity; taking into account conservative substitutions, -40% of the residues are similar. The E. coli Na+/proline cotransporter is 24% smaller than the human Na+/glucose cotransporter (502 vs. 664 residues). This cotransporter lacks the C-terminal region (residues 593-664), including part of the highly charged region near the C terminus (567-631). Except for segments 3 and 4, the predicted membrane-spanning sequences for the Na+/ proline transporter have positions very similar to those predicted for the Na+/glucose cotransporter. The Na+/ proline cotransporter has shorter hydrophilic segments between the membrane-spanning segments. Membrane segment 11 may represent a non-membrane-spanning region, owing to its short length and/or high hydrophobic moment, and this region corresponds to the hydrophobic surface region 9a of the Na+/glucose cotransporter. The Na+/ glucose transporter model in Fig. 3 shows regions of particular sequence homology to the Na+/proline transporter as thick line segments. The transporters have two charged regions in common at residues 130-140 and 408-420. The similarity is further emphasized when comparing the secondary structural elements (19), such as the occurrence of an a-helix in front of membrane-spanning regions 4. Earlier we showed that a tyrosine residue is located at or near the Na+-binding site of the rabbit brush border Na+/ glucose and Na+/proline cotransporter (8, 20). Of the 25 Na+/glucose cotransporter tyrosine residues, only 131, 191, 410, and 528 are located in the regions conserved with the E. coli proline carrier (Fig. 3). One or more of these may be involved in Na+ binding. If lysine residues are involved in glucose and proline binding, only three ofthese (residues 415, 420, and 549) are conserved in the rabbit, human, and E. coli transporters. In the case of the putP proline transporter, experiments with mutants (21) suggest that Arg-257 is related to the structure of the Na+ binding and Na+ coupling sites. This residue is conserved in the Na+/glucose cotransporter
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09 29
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FIG. 3. Proposed model of the membrane orientation of the human intestinal Na+/glucose cotransporter. The method of Eisenberg et al. (9) was used with windows of 21 and 11 amino acids to predict membrane-spanning segments 1-11. Segment 7a is shown with dotted lines and represents an additional possible membrane-spanning segment. CHO indicates an N-glycosylation site. The region labeled Sur is a surface-seeking membrane region and was predicted using the Eisenberg method. Heavy lines in the sequence indicate regions of particular sequence homology with the E. coli Na+/proline transporter (4). Clusters of negative and positive charges in the hydrophilic regions of the sequence are represented as - and +.
~~~~
Proc. Nati. Acad. Sci. USA 86 (1989)
Biochemistry: Hediger et al.
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Biochemistry: Hediger et al.
plasma membranes; for example, the four conserved tyrosines (131, 191, 410, and 528) may be involved in Na' binding. The structural and functional similarity between the human Na+/glucose cotransporter and the E. coli Na+/proline cotransporter is certainly too extensive to have arisen by convergent evolution. An example of convergent evolution is evident in the Na+/glucose and Na+/melibiose cotransporters where there is no detectible similarity at either the DNA or amino acid levels. The homology of the Na+/glucose to the Na+/proline cotransporter suggests, therefore, that Na'cotransport proteins, or an ancestor thereof, were present in organisms before the divergence of prokaryotes and eukaryotes. We thank Hsin-Shung Lee for carrying out oocyte experiments, B. Blumberg for advice on the evaluation of the significance of the sequence homology, and M. Stelzner and E. W. Fonkalsrud for providing human ileum to prepare the cDNA library. This work was supported by grants from the U.S. Public Health Service (DK 19567 and DK 17328). 1. Kaback, H. R. (1986) Annu. Rev. Biophys. Chem. 15, 279-319. 2. Hopfer, U. (1987) in Physiology of the Gastrointestinal Tract, ed. Johnson, L. R. (Raven, New York), pp. 1499-1526. 3. Yazyu, H., Shiota-Niiya, S., Shimamoto, T., Kanazawa, H., Futai, M. & Tsuchiya, T. (1984) J. Biol. Chem. 259, 4320-4326. 4. Nakao, T., Yamato, I. & Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75. 5. Gubler, U. & Hoffman, B. (1983) Gene 25, 263-269. 6. Hediger, M. A., Coady, M. J., Ikeda, T. S. & Wright, E. M. (1987) Nature (London) 330, 379-381. 7. Peerce, B. E. & Wright, E. M. (1984) J. Biol. Chem. 259, 14105-14112.
Proc. Nadl. Acad. Sci. USA 86 (1989) 8. Peerce, B. E. & Wright, E. M. (1985) J. Biol. Chem. 260, 6026-6031. 9. Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R. (1984) J. Mol. Biol. 179, 125-142. 10. Mendlein, J., Hediger, M., Coady, M. & Wright, E. M. (1988) FASEB J. 2, A1021. 11. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448. 12. Devereaux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395. 13. Hanada, K., Yamato, I. & Anraku, Y. (1988) J. Biol. Chem. 263, 7181-7185. 14. Lipman, D. J. & Pearson, W. R. (1985) Science 227, 14351441. 15. Kanehisa, M. I. (1982) Nucleic Acids Res. 10, 183-196. 16. Dayhoff, M. O., Barker, W. C. & Hunt, L. T. (1983) Methods Enzymol 91, 524-545. 17. Doolittle, R. F. (1981) Science 214, 149-159. 18. Maiden, M. C. J., Davis, E. O., Baldwin, S. A., Moore, D. C. M. & Henderson, P. J. F. (1987) Nature (London) 325, 641-643. 19. Chou, P. Y. & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 251-276. 20. Wright, E. M. & Peerce, B. E. (1984) J. Biol. Chem. 259, 14993-14996. 21. Ohsawa, M., Mogi, T., Yamamoto, H., Yamato, I. & Anraku, Y. (1988) J. Bacteriol 170, 5185-5191. 22. Stewart, L. M. D. & Booth, I. R. (1983) FEMS Microbiol. Lett. 19, 161-164. 23. Chen, C.-C., Tsuchiya, T., Yamane, Y., Wood, J. M. & Wilson, T. H. (1985) J. Mol. Biol. 84, 157-164. 24. Chen, C.-C. & Wilson, T. H. (1986) J. Biol. Chem. 261, 2599-2604.
