© The Rockefeller University Press,
0021-9525/1997//1239 $5.00
The Journal of Cell Biology, Volume 136, Number 6,
, 1997 1239-1247
Molecular Cloning and Functional Characterization of the Receptor for Clostridium perfringens Enterotoxin
Jun Katahira*,
Norimitsu Inoue
,
Yasuhiko Horiguchi*,
Morihiro Matsuda*, and
Nakaba Sugimoto*
* Department of Bacterial Toxinology and
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan
A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and poreformation in the cell membrane.
Abbreviations used in this paper: CPE, Clostridium perfringens enterotoxin; CPE-R, CPE-receptor gene; H10PER, histidine-tagged CPE COOHteminal fragment; PE, phycoerythrin.
We are indebted to Dr. T. Kinoshita and members of the Department of Immunoregulation for their helpful discussions and providing plasmids. We are grateful to the following persons of Research Institute for Microbial Diseases, Osaka University; Dr. H. Nojima (Department of Molecular Genetics) for helpful suggestions on library construction and Drs. N. Wakamiya (Department of Viral Infections) and K. Nagayama (Department of Bacterial Infections) for providing cell lines. We are also grateful to Dr. T. Asao (Osaka Prefectural Institute of Public Health) for providing the C. perfringens strain. We also thank K. Nakamura, Central Laboratory, Research Institute for Microbial Diseases, Osaka University, for cell sorting. We are grateful to Dr. S. Kozaki (University of Osaka Prefecture) for critical reading of this manuscript.
Please address all correspondence to Jun Katahira, Department of Bacterial Toxicology, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan. Tel.: 81-6-879-8285; Fax.: 81-6-879-8283; E-mail: katahira{at}biken.osaka-u.ac.jp

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