Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Jan;176(1):149–156. doi: 10.1128/jb.176.1.149-156.1994

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

O Matsushita 1, K Yoshihara 1, S Katayama 1, J Minami 1, A Okabe 1
PMCID: PMC205026  PMID: 8282691

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.

Full text

PDF
149

Images in this article

152Image
on p.152
152Image
on p.152
154Image
on p.154

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Birkedal-Hansen H. Catabolism and turnover of collagens: collagenases. Methods Enzymol. 1987;144:140–171. doi: 10.1016/0076-6879(87)44177-3. [DOI] [PubMed] [Google Scholar]
  3. Birkedal-Hansen H., Taylor R. E. Detergent-activation of latent collagenase and resolution of its component molecules. Biochem Biophys Res Commun. 1982 Aug 31;107(4):1173–1178. doi: 10.1016/s0006-291x(82)80120-4. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bond M. D., Van Wart H. E. Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry. 1984 Jun 19;23(13):3085–3091. doi: 10.1021/bi00308a036. [DOI] [PubMed] [Google Scholar]
  6. Bond M. D., Van Wart H. E. Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography. Biochemistry. 1984 Jun 19;23(13):3077–3085. doi: 10.1021/bi00308a035. [DOI] [PubMed] [Google Scholar]
  7. Bond M. D., Van Wart H. E. Relationship between the individual collagenases of Clostridium histolyticum: evidence for evolution by gene duplication. Biochemistry. 1984 Jun 19;23(13):3092–3099. doi: 10.1021/bi00308a037. [DOI] [PubMed] [Google Scholar]
  8. Canard B., Saint-Joanis B., Cole S. T. Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens. Mol Microbiol. 1992 Jun;6(11):1421–1429. doi: 10.1111/j.1365-2958.1992.tb00862.x. [DOI] [PubMed] [Google Scholar]
  9. Garnier T., Canard B., Cole S. T. Cloning, mapping, and molecular characterization of the rRNA operons of Clostridium perfringens. J Bacteriol. 1991 Sep;173(17):5431–5438. doi: 10.1128/jb.173.17.5431-5438.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hatheway C. L. Toxigenic clostridia. Clin Microbiol Rev. 1990 Jan;3(1):66–98. doi: 10.1128/cmr.3.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kameyama S., Akama K. Purification and some properties of kappa toxin of Clostridium perfringens. Jpn J Med Sci Biol. 1971 Feb;24(1):9–23. doi: 10.7883/yoken1952.24.9. [DOI] [PubMed] [Google Scholar]
  12. Karn J., Matthes H. W., Gait M. J., Brenner S. A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI. Gene. 1984 Dec;32(1-2):217–224. doi: 10.1016/0378-1119(84)90049-0. [DOI] [PubMed] [Google Scholar]
  13. Katayama S., Matsushita O., Minami J., Mizobuchi S., Okabe A. Comparison of the alpha-toxin genes of Clostridium perfringens type A and C strains: evidence for extragenic regulation of transcription. Infect Immun. 1993 Feb;61(2):457–463. doi: 10.1128/iai.61.2.457-463.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kato T., Takahashi N., Kuramitsu H. K. Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene, which expresses a novel collagenase activity. J Bacteriol. 1992 Jun;174(12):3889–3895. doi: 10.1128/jb.174.12.3889-3895.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keil B. Some newly characterized collagenases from procaryotes and lower eucaryotes. Mol Cell Biochem. 1979 Jan 26;23(2):87–108. doi: 10.1007/BF00226230. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lepage T., Gache C. Early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo. EMBO J. 1990 Sep;9(9):3003–3012. doi: 10.1002/j.1460-2075.1990.tb07493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lwebuga-Mukasa J. S., Harper E., Taylor P. Collagenase enzymes from Clostridium: characterization of individual enzymes. Biochemistry. 1976 Oct 19;15(21):4736–4741. doi: 10.1021/bi00666a031. [DOI] [PubMed] [Google Scholar]
  19. Merkel J. R., Dreisbach J. H. Purification and characterization of a marine bacterial collagenase. Biochemistry. 1978 Jul 11;17(14):2857–2863. doi: 10.1021/bi00607a025. [DOI] [PubMed] [Google Scholar]
  20. Mookhtiar K. A., Steinbrink D. R., Van Wart H. E. Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities. Biochemistry. 1985 Nov 5;24(23):6527–6533. doi: 10.1021/bi00344a033. [DOI] [PubMed] [Google Scholar]
  21. Ohara T., Makino K., Shinagawa H., Nakata A., Norioka S., Sakiyama F. Cloning, nucleotide sequence, and expression of Achromobacter protease I gene. J Biol Chem. 1989 Dec 5;264(34):20625–20631. [PubMed] [Google Scholar]
  22. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rood J. I., Cole S. T. Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol Rev. 1991 Dec;55(4):621–648. doi: 10.1128/mr.55.4.621-648.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  26. Takeuchi H., Shibano Y., Morihara K., Fukushima J., Inami S., Keil B., Gilles A. M., Kawamoto S., Okuda K. Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase. Biochem J. 1992 Feb 1;281(Pt 3):703–708. doi: 10.1042/bj2810703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Wart H. E., Steinbrink D. R. Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum. Biochemistry. 1985 Nov 5;24(23):6520–6526. doi: 10.1021/bi00344a032. [DOI] [PubMed] [Google Scholar]
  28. WUENSCH E., HEIDRICH H. G. ZUR QUANTITATIVEN BESTIMMUNG DER KOLLAGENASE. Hoppe Seylers Z Physiol Chem. 1963;333:149–151. doi: 10.1515/bchm2.1963.333.1.149. [DOI] [PubMed] [Google Scholar]
  29. Wilson M. J., Strasser M., Vogel M. M., Sinha A. A. Calcium-dependent and calcium-independent gelatinolytic proteinase activities of the rat ventral prostate and its secretion: characterization and effect of castration and testosterone treatment. Biol Reprod. 1991 May;44(5):776–785. doi: 10.1095/biolreprod44.5.776. [DOI] [PubMed] [Google Scholar]
  30. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wolf U., Bauer D., Traub W. H. Collagenase of Clostridium perfringens type A: degradation of human complement component C1q. Zentralbl Bakteriol. 1991 Dec;276(1):27–35. doi: 10.1016/s0934-8840(11)80215-x. [DOI] [PubMed] [Google Scholar]
  32. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES