Localization of the receptor-binding region of Clostridium perfringens enterotoxin utilizing cloned toxin fragments and synthetic peptides. The 30 C-terminal amino acids define a functional binding region.
In this study a short sequence encoding the receptor-binding activity of the much larger 35-kDa enterotoxin elaborated by Clostridium perfringens was localized by recombinant DNA techniques. Defined fragments corresponding to portions of the enterotoxin gene were cloned into an Escherichia coli expression vector system, and these lysates were analyzed for their ability to compete for binding with native C. perfringens enterotoxin (CPE). The lysate containing CPE290-319 (CPE sequence encompassing residues 290-319) was shown to compete with 125I-CPE for specific binding sites on rabbit intestinal brush border membranes. To confirm this finding, a peptide corresponding to the CPE amino acid sequence 290-319 was synthesized and found to completely block CPE specific binding. To demonstrate directly that CPE290-319 can act as a competitive antagonist of CPE cytotoxicity for physiologic receptors, Vero cells were preincubated with either E. coli lysates containing CPE290-319 or the synthetic peptide corresponding to this sequence. Preincubation of Vero cells with either the lysate or the peptide completely protected these cells from CPE challenge. This information localizes the C-terminal 30 residues of CPE (CPE290-319) as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor.
Hanna
P C
PC
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Mietzner
T A
TA
Schoolnik
G K
GK
McClane
B A
BA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006162:Guanylate Cyclase; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide
D004269:DNA, Bacterial; D004768:Enterotoxins; D015345:Oligonucleotide Probes; D010455:Peptides; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C035598:enterotoxin, Clostridium; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled