Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides.
The effect of lambda-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inactive epsilon-prototoxin produced by the same organism was examined. When the purified epsilon-prototoxin was incubated with the purified lambda-toxin at 37 C for 2 hr, the 32.5-kDa epsilon-prototoxin was processed into a 30.5-kDa polypeptide, as determined by SDS-polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without lambda-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by lambda-toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The epsilon-toxin gene was cloned and sequenced. Determination of the N-terminal amino acid sequence of each activated epsilon-prototoxin revealed that lambda-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated epsilon-prototoxin was also determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The C-terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N-terminal but also C-terminal peptide is responsible for activation of the prototoxin.
Minami
J
J
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Japan.
Katayama
S
S
Matsushita
O
O
Matsushita
C
C
Okabe
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D001681:Biological Assay; D002918:Chymotrypsin / Q000494:pharmacology; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D017077:Culture Media, Conditioned; D007928:Lethal Dose 50; D008297:Male; D008666:Metalloendopeptidases / Q000494:pharmacology*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000737:chemistry; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology; D014774:Virulence
D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D017077:Culture Media, Conditioned; D011498:Protein Precursors; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C030895:caseinase