Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease.
The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.
Jin
F
F
Department of Microbiology, Kagawa Medical School, Japan.
Matsushita
O
O
Katayama
S
S
Jin
S
S
Matsushita
C
C
Minami
J
J
Okabe
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000737:chemistry / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D003094:Collagen / Q000378:metabolism; D003238:Connective Tissue / Q000473:pathology; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008297:Male; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011480:Protease Inhibitors / Q000494:pharmacology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000473:pathology; D013379:Substrate Specificity
D001798:Blood Proteins; D011480:Protease Inhibitors; D003094:Collagen; D008666:Metalloendopeptidases; C030895:caseinase