Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase.
The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.
Department of Microbiology, Kagawa Medical School, Japan.
K
K
Yoshihara
O
O
Matsushita
J
J
Minami
A
A
Okabe
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D017364:Collagenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018093:Gelatinases / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D015183:Restriction Mapping; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D017364:Collagenases; D018093:Gelatinases