Clostridium perfringens Enterotoxin Utilizes Two Structurally Related Membrane Proteins as Functional Receptors in Vivo

Human and mouse cDNAs showing homology to theClostridium perfringens enterotoxin (CPE) receptor gene (CPE-R) from Vero cells (DDBJ/EMBL/GenBankTMaccession no. D88492) (Katahira, J., Inoue, N., Horiguchi, Y., Matsuda, M., and Sugimoto, N. (1997) J. Cell Biol. 136, 1239–1247) were cloned. They were classified into two groups, the Vero cell CPE receptor homologues and rat androgen withdrawal apoptosis protein (RVP1; accession no. M74067) homologues, based on the similarities of primary amino acid sequences. L929 cells that were originally insensitive to CPE became sensitive to CPE on their transfection with cDNAs encoding either the CPE receptor or RVP1 homologues, indicating that these gene products are not only structurally similar but also functionally active as receptors for CPE. By binding assay, the human RVP1 homologue showed differences in affinity and capacity of binding from those of the human CPE receptor. Northern blot analysis showed that mouse homologues of the CPE receptor and RVP1 are expressed abundantly in mouse small intestine. The expression ofCPE-R mRNA in the small intestine was restricted to cryptic enterocytes, indicating that the CPE receptor is expressed in intestinal epithelial cells. These results are consistent with reports that CPE binds to the small intestinal cells via two different kinds of receptors. High levels of expression of CPE-R and/orRVP1 mRNA were also detected in other organs, including the lungs, liver, and kidneys, but only low levels were expressed in heart and skeletal muscles. These results indicate that CPE uses structurally related cellular proteins as functional receptors in vivo and that organs that have not so far been recognized as CPE-sensitive have the potential to be targets of CPE. Human and mouse cDNAs showing homology to theClostridium perfringens enterotoxin (CPE) receptor gene (CPE-R) from Vero cells (DDBJ/EMBL/GenBankTMaccession no. D88492) (Katahira, J., Inoue, N., Horiguchi, Y., Matsuda, M., and Sugimoto, N. (1997) J. Cell Biol. 136, 1239–1247) were cloned. They were classified into two groups, the Vero cell CPE receptor homologues and rat androgen withdrawal apoptosis protein (RVP1; accession no. M74067) homologues, based on the similarities of primary amino acid sequences. L929 cells that were originally insensitive to CPE became sensitive to CPE on their transfection with cDNAs encoding either the CPE receptor or RVP1 homologues, indicating that these gene products are not only structurally similar but also functionally active as receptors for CPE. By binding assay, the human RVP1 homologue showed differences in affinity and capacity of binding from those of the human CPE receptor. Northern blot analysis showed that mouse homologues of the CPE receptor and RVP1 are expressed abundantly in mouse small intestine. The expression ofCPE-R mRNA in the small intestine was restricted to cryptic enterocytes, indicating that the CPE receptor is expressed in intestinal epithelial cells. These results are consistent with reports that CPE binds to the small intestinal cells via two different kinds of receptors. High levels of expression of CPE-R and/orRVP1 mRNA were also detected in other organs, including the lungs, liver, and kidneys, but only low levels were expressed in heart and skeletal muscles. These results indicate that CPE uses structurally related cellular proteins as functional receptors in vivo and that organs that have not so far been recognized as CPE-sensitive have the potential to be targets of CPE. The enterotoxin produced by Clostridium perfringens(CPE) 1The abbreviations used are: CPE, C. perfringens enterotoxin; EST, expressed sequence tag; ORF, open reading frame; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; PBS, phosphate-buffered saline; kb, kilobase pair(s). 1The abbreviations used are: CPE, C. perfringens enterotoxin; EST, expressed sequence tag; ORF, open reading frame; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; PBS, phosphate-buffered saline; kb, kilobase pair(s). is a simple protein with a molecular weight of ∼35,000. Known as a causative agent of diarrhea, this organism (1Stark R.L. Duncan C.L. Infect. Immun. 1971; 4: 89-96Crossref PubMed Google Scholar) elicits fluid accumulation in the intestinal tract by altering the membrane permeability of intestinal epithelial cells (2McDonel J.L. Duncan C.L. Infect. Immun. 1975; 12: 1214-1218Crossref PubMed Google Scholar, 3McDonel J.L. Chang L.W. Pounds J.G. Duncan C.L. Lab. Invest. 1978; 39: 210-217PubMed Google Scholar). Pore formation in the cytoplasmic membrane is now accepted as the underlying mechanism of its effect (4Sugimoto N. Takagi M. Ozutsumi K. Harada S. Matsuda M. Biochem. Biophys. Res. Commun. 1988; 156: 551-556Crossref PubMed Scopus (21) Google Scholar, 5Matsuda M. Ozutsumi K. Iwahashi H. Sugimoto N. Biochem. Biophys. Res. Commun. 1986; 141: 704-710Crossref PubMed Scopus (39) Google Scholar, 6Sugimoto N. Ozutsumi K. Matsuda M. Eur. J. Epidemiol. 1985; 1: 264-273PubMed Google Scholar, 7McClane B.A. McDonel J.L. Biochim. Biophys. Acta. 1981; 641: 401-409Crossref PubMed Scopus (35) Google Scholar).Not only humans, but also various experimental animals have been shown to be sensitive to CPE (8McDonel J.L. Dorner F. Drews H. Pharmacology of Bacterial Toxins. Pergamon Press, Oxford1986: 477-517Google Scholar), suggesting that the sensitivity is not restricted to a particular species. Although the natural target of CPE is the intestine, CPE has also been detected in other tissues and organs, including the liver and kidneys, after its intravenous injection into rats and mice (9Skjelkvale R. Tolleshaug H. Jarmund T. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. Immnnol. 1980; 88: 95-102Google Scholar). In addition, cultured cells of the intestine, liver, and kidneys from various species have been shown to be sensitive to CPE (10McDonel J.L. Biochemistry. 1980; 19: 4801-4807Crossref PubMed Scopus (40) Google Scholar, 11Tolleshaug H. Skjelkvale R. Berg T. Infect. Immun. 1982; 37: 486-491Crossref PubMed Google Scholar, 12McClane B.A. McDonel J.L. J. Cell. Physiol. 1979; 99: 191-199Crossref PubMed Scopus (66) Google Scholar, 13Matsuda M. Sugimoto N. Biochem. Biophys. Res. Commun. 1979; 91: 629-636Crossref PubMed Scopus (50) Google Scholar, 14Horiguchi Y. Uemura T. Kozaki S. Sakaguchi G. FEMS Microbiol. Lett. 1985; 28: 131-135Crossref Google Scholar). Two different receptors with high and low affinity to CPE have been found on the surface of rabbit intestinal epithelial cells (10McDonel J.L. Biochemistry. 1980; 19: 4801-4807Crossref PubMed Scopus (40) Google Scholar). Horiguchi et al. (14Horiguchi Y. Uemura T. Kozaki S. Sakaguchi G. FEMS Microbiol. Lett. 1985; 28: 131-135Crossref Google Scholar) showed that Vero cells and Madin-Darby canine kidney cells, both of which are derived from kidneys, express high and low affinity receptors, respectively. Since the cytotoxic action of CPE to target cells requires its binding to specific receptors (14Horiguchi Y. Uemura T. Kozaki S. Sakaguchi G. FEMS Microbiol. Lett. 1985; 28: 131-135Crossref Google Scholar, 15McDonel J.L. McClane B.A. Biochem. Biophys. Res. Commun. 1979; 87: 497-504Crossref PubMed Scopus (40) Google Scholar), at least two molecules with different affinities to CPE are considered to exist in various organs of a wide range of species.Recently, we cloned a cDNA for the CPE receptor (CPE-R) from a CPE-sensitive Vero cell cDNA library (16Katahira J. Inoue N. Horiguchi Y. Matsuda M. Sugimoto N. J. Cell Biol. 1997; 136: 1239-1247Crossref PubMed Scopus (239) Google Scholar). The cDNA encodes a highly hydrophobic transmembrane protein of ∼22 kDa, the physiological functions of which have not yet been elucidated. The amino acid sequence of the Vero cell CPE receptor showed close similarity to that of the rat androgen withdrawal apoptosis protein RVP1 (17Briehl M.M. Miesfeld R.L. Mol. Endocrinol. 1991; 5: 1381-1388Crossref PubMed Scopus (112) Google Scholar). The Vero cell CPE receptor corresponds to the reported high affinity binding site for CPE. CPE-R was found to be expressed in CPE-sensitive cell lines from different origins;i.e. Vero cells from monkey kidneys, Henle intestine 407 cells from human small intestine, and Hep3B cells from human liver. In contrast to these sensitive cells, CPE-insensitive cells, such as the human erythroleukemia cell line K562 and human lymphoblastoid cell line JY, were found not to express CPE-R, suggesting that the expression of CPE-R is tissue- and/or organ-specific.In this study, to obtain further insight into the receptors for CPEin vivo, we sought for human and mouse cDNAs similar to the Vero cell CPE-R. Here we report that these gene products can be classified into two groups homologous to the CPE receptor and RVP1, based on their similarities in structure to the Vero cell CPE receptor and RVP1 and differences in their affinities and binding capacity to CPE, and that they both have the ability to confer CPE sensitivity to the insensitive L929 cell line. We also found that the expressions of CPE-R and RVP1 were observed in a wide variety of organs, including the small intestine. These data indicate that two different gene products are expressed in target organs in vivo and are probably recognized as different affinity receptors by CPE. The enterotoxin produced by Clostridium perfringens(CPE) 1The abbreviations used are: CPE, C. perfringens enterotoxin; EST, expressed sequence tag; ORF, open reading frame; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; PBS, phosphate-buffered saline; kb, kilobase pair(s). 1The abbreviations used are: CPE, C. perfringens enterotoxin; EST, expressed sequence tag; ORF, open reading frame; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; PBS, phosphate-buffered saline; kb, kilobase pair(s). is a simple protein with a molecular weight of ∼35,000. Known as a causative agent of diarrhea, this organism (1Stark R.L. Duncan C.L. Infect. Immun. 1971; 4: 89-96Crossref PubMed Google Scholar) elicits fluid accumulation in the intestinal tract by altering the membrane permeability of intestinal epithelial cells (2McDonel J.L. Duncan C.L. Infect. Immun. 1975; 12: 1214-1218Crossref PubMed Google Scholar, 3McDonel J.L. Chang L.W. Pounds J.G. Duncan C.L. Lab. Invest. 1978; 39: 210-217PubMed Google Scholar). Pore formation in the cytoplasmic membrane is now accepted as the underlying mechanism of its effect (4Sugimoto N. Takagi M. Ozutsumi K. Harada S. Matsuda M. Biochem. Biophys. Res. Commun. 1988; 156: 551-556Crossref PubMed Scopus (21) Google Scholar, 5Matsuda M. Ozutsumi K. Iwahashi H. Sugimoto N. Biochem. Biophys. Res. Commun. 1986; 141: 704-710Crossref PubMed Scopus (39) Google Scholar, 6Sugimoto N. Ozutsumi K. Matsuda M. Eur. J. Epidemiol. 1985; 1: 264-273PubMed Google Scholar, 7McClane B.A. McDonel J.L. Biochim. Biophys. Acta. 1981; 641: 401-409Crossref PubMed Scopus (35) Google Scholar). Not only humans, but also various experimental animals have been shown to be sensitive to CPE (8McDonel J.L. Dorner F. Drews H. Pharmacology of Bacterial Toxins. Pergamon Press, Oxford1986: 477-517Google Scholar), suggesting that the sensitivity is not restricted to a particular species. Although the natural target of CPE is the intestine, CPE has also been detected in other tissues and organs, including the liver and kidneys, after its intravenous injection into rats and mice (9Skjelkvale R. Tolleshaug H. Jarmund T. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. Immnnol. 1980; 88: 95-102Google Scholar). In addition, cultured cells of the intestine, liver, and kidneys from various species have been shown to be sensitive to CPE (10McDonel J.L. Biochemistry. 1980; 19: 4801-4807Crossref PubMed Scopus (40) Google Scholar, 11Tolleshaug H. Skjelkvale R. Berg T. Infect. Immun. 1982; 37: 486-491Crossref PubMed Google Scholar, 12McClane B.A. McDonel J.L. J. Cell. Physiol. 1979; 99: 191-199Crossref PubMed Scopus (66) Google Scholar, 13Matsuda M. Sugimoto N. Biochem. Biophys. Res. Commun. 1979; 91: 629-636Crossref PubMed Scopus (50) Google Scholar, 14Horiguchi Y. Uemura T. Kozaki S. Sakaguchi G. FEMS Microbiol. Lett. 1985; 28: 131-135Crossref Google Scholar). Two different receptors with high and low affinity to CPE have been found on the surface of rabbit intestinal epithelial cells (10McDonel J.L. Biochemistry. 1980; 19: 4801-4807Crossref PubMed Scopus (40) Google Scholar). Horiguchi et al. (14Horiguchi Y. Uemura T. Kozaki S. Sakaguchi G. FEMS Microbiol. Lett. 1985; 28: 131-135Crossref Google Scholar) showed that Vero cells and Madin-Darby canine kidney cells, both of which are derived from kidneys, express high and low affinity receptors, respectively. Since the cytotoxic action of CPE to target cells requires its binding to specific receptors (14Horiguchi Y. Uemura T. Kozaki S. Sakaguchi G. FEMS Microbiol. Lett. 1985; 28: 131-135Crossref Google Scholar, 15McDonel J.L. McClane B.A. Biochem. Biophys. Res. Commun. 1979; 87: 497-504Crossref PubMed Scopus (40) Google Scholar), at least two molecules with different affinities to CPE are considered to exist in various organs of a wide range of species. Recently, we cloned a cDNA for the CPE receptor (CPE-R) from a CPE-sensitive Vero cell cDNA library (16Katahira J. Inoue N. Horiguchi Y. Matsuda M. Sugimoto N. J. Cell Biol. 1997; 136: 1239-1247Crossref PubMed Scopus (239) Google Scholar). The cDNA encodes a highly hydrophobic transmembrane protein of ∼22 kDa, the physiological functions of which have not yet been elucidated. The amino acid sequence of the Vero cell CPE receptor showed close similarity to that of the rat androgen withdrawal apoptosis protein RVP1 (17Briehl M.M. Miesfeld R.L. Mol. Endocrinol. 1991; 5: 1381-1388Crossref PubMed Scopus (112) Google Scholar). The Vero cell CPE receptor corresponds to the reported high affinity binding site for CPE. CPE-R was found to be expressed in CPE-sensitive cell lines from different origins;i.e. Vero cells from monkey kidneys, Henle intestine 407 cells from human small intestine, and Hep3B cells from human liver. In contrast to these sensitive cells, CPE-insensitive cells, such as the human erythroleukemia cell line K562 and human lymphoblastoid cell line JY, were found not to express CPE-R, suggesting that the expression of CPE-R is tissue- and/or organ-specific. In this study, to obtain further insight into the receptors for CPEin vivo, we sought for human and mouse cDNAs similar to the Vero cell CPE-R. Here we report that these gene products can be classified into two groups homologous to the CPE receptor and RVP1, based on their similarities in structure to the Vero cell CPE receptor and RVP1 and differences in their affinities and binding capacity to CPE, and that they both have the ability to confer CPE sensitivity to the insensitive L929 cell line. We also found that the expressions of CPE-R and RVP1 were observed in a wide variety of organs, including the small intestine. These data indicate that two different gene products are expressed in target organs in vivo and are probably recognized as different affinity receptors by CPE. We thank members in the Departments of Bacterial Toxinology and Immunoregulation for critical comments and help during this work. We also thank Dr. Y. Kameoka (Division of Genetic Resources, National Institute of Health) for valuable suggestions.