Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene

Abstract

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.

The genome sequence of Clostridium tetani , the causative agent of tetanus disease

“…Interestingly, pE88 encodes an additional virulence factor, a 114-kDa collagenase, designated ColT (CTP33), an enzyme that may play an important role in C. tetani pathogenesis because of its activity to destroy tissue integrity in the infected host (20). A multiple sequence alignment with other known clostridial collagenases of Clostridium histolyticum (21), C. perfringens (22) and the one encoded on the genome of C. botulinum (Sanger Centre, Cambridge, UK, C. botulinum sequencing project), designated ColB, revealed a different domain structure: ColT lacks segment 2, the so-called PKD domain of unknown function (Fig. 2).…”

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

“…DNA fragments for gene probes were labelled with an AlkPhos-direct kit (Amersham Pharmacia Biotech), and signals were detected by CDPstar chemiluminescence. The colA, plc, pfoA and luxS probes were prepared by polymerase chain reaction (PCR) from pKY3135 (Matsushita et al, 1994), pKB300 , pTS310 (Shimizu et al, 1991) and pSB235 (Banu et al, 2000), respectively, using the appropriate primer sets. The relative amount of mRNA was quantified by densitometry (NIH-Image; http://rsb.info.nih.gov/nih-image/).…”

Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

“…Metallo-collagenases, first discovered in tadpole tissue explants (Gross and Lapiere, 1962), are zinc-containing enzymes that also generally require calcium for their optimum activity and stability, and cleave the collagen helix at a specific locus under physiological conditions (Gawston and Murphy, 1981;Harris and Vater, 1982;Sellers and Murphy, 1981;Stricklin et al, 1977). These enzymes have been widely studied from various mammalian tissues (Harris and Vater, 1982;Sellers and Murphy, 1981) as well as from bacteria, such as Bacillus cereus (Makinen and Makinen, 1987), Clostridium histolyticum (Bond and Van Wart, 1984a, b;Peterkofsky, 1982), Achromobacter (Nguyen et al, 1988), Vibrio alginolyticus (Takeuchi et al, 1992) and Clostridium perfringens (Matsuhita et al, 1994), and snake venom (Bjarnason and Fox, 1994). These metallo-collagenases, extracellular enzymes, are involved in remodeling the extracellular matrix.…”