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. 1993 Oct;61(10):4099–4104. doi: 10.1128/iai.61.10.4099-4104.1993

Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus.

M Monod 1, S Paris 1, D Sanglard 1, K Jaton-Ogay 1, J Bille 1, J P Latgé 1
PMCID: PMC281130  PMID: 8406798

Abstract

A metalloprotease (MEP) secreted by Aspergillus fumigatus was isolated from an alkaline protease-deficient mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxy-methyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fumigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.

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Selected References

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  1. Bodey G. P., Vartivarian S. Aspergillosis. Eur J Clin Microbiol Infect Dis. 1989 May;8(5):413–437. doi: 10.1007/BF01964057. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Denning D. W., Ward P. N., Fenelon L. E., Benbow E. W. Lack of vessel wall elastolysis in human invasive pulmonary aspergillosis. Infect Immun. 1992 Dec;60(12):5153–5156. doi: 10.1128/iai.60.12.5153-5156.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Frosco M., Chase T., Jr, Macmillan J. D. Purification and properties of the elastase from Aspergillus fumigatus. Infect Immun. 1992 Mar;60(3):728–734. doi: 10.1128/iai.60.3.728-734.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jaton-Ogay K., Suter M., Crameri R., Falchetto R., Fatih A., Monod M. Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus. FEMS Microbiol Lett. 1992 Apr 15;71(2):163–168. doi: 10.1016/0378-1097(92)90506-j. [DOI] [PubMed] [Google Scholar]
  6. Kothary M. H., Chase T., Jr, Macmillan J. D. Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect Immun. 1984 Jan;43(1):320–325. doi: 10.1128/iai.43.1.320-325.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kurup V. P., Kumar A. Immunodiagnosis of aspergillosis. Clin Microbiol Rev. 1991 Oct;4(4):439–456. doi: 10.1128/cmr.4.4.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Latgé J. P., Moutaouakil M., Debeaupuis J. P., Bouchara J. P., Haynes K., Prévost M. C. The 18-kilodalton antigen secreted by Aspergillus fumigatus. Infect Immun. 1991 Aug;59(8):2586–2594. doi: 10.1128/iai.59.8.2586-2594.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Monod M., Paris S., Sarfati J., Jaton-Ogay K., Ave P., Latgé J. P. Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus. FEMS Microbiol Lett. 1993 Jan 1;106(1):39–46. doi: 10.1111/j.1574-6968.1993.tb05932.x. [DOI] [PubMed] [Google Scholar]
  11. Monod M., Togni G., Rahalison L., Frenk E. Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus. J Med Microbiol. 1991 Jul;35(1):23–28. doi: 10.1099/00222615-35-1-23. [DOI] [PubMed] [Google Scholar]
  12. Panneerselvam M., Dhar S. C. Chromatographic purification and homogeneity of extracellular acid proteinase of Aspergillus Fumigatus. Ital J Biochem. 1980 Mar-Apr;29(2):102–112. [PubMed] [Google Scholar]
  13. Panneerselvam M., Dhar S. C. Physico-chemical properties of the acid proteinase from A. fumigatus. Ital J Biochem. 1981 Jan-Feb;30(1):63–74. [PubMed] [Google Scholar]
  14. Reichard U., Büttner S., Eiffert H., Staib F., Rüchel R. Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. J Med Microbiol. 1990 Dec;33(4):243–251. doi: 10.1099/00222615-33-4-243. [DOI] [PubMed] [Google Scholar]
  15. Rhodes J. C., Amlung T. W., Miller M. S. Isolation and characterization of an elastinolytic proteinase from Aspergillus flavus. Infect Immun. 1990 Aug;58(8):2529–2534. doi: 10.1128/iai.58.8.2529-2534.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rhodes J. C., Amlung T. W. The elastinolytic proteinase of Aspergillus flavus is not glycosylated. J Med Vet Mycol. 1991;29(6):407–411. [PubMed] [Google Scholar]
  17. Staib F. Extracellular proteolytic activity of Aspergillus fumigatus strains with septum-like structures in their phialides in serum-albumin agar--preliminary report. Zentralbl Bakteriol Mikrobiol Hyg A. 1982 Jun;252(2):279–285. [PubMed] [Google Scholar]
  18. Tang C. M., Cohen J., Holden D. W. An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity. Mol Microbiol. 1992 Jun;6(12):1663–1671. doi: 10.1111/j.1365-2958.1992.tb00891.x. [DOI] [PubMed] [Google Scholar]
  19. Tang C. M., Cohen J., Krausz T., Van Noorden S., Holden D. W. The alkaline protease of Aspergillus fumigatus is not a virulence determinant in two murine models of invasive pulmonary aspergillosis. Infect Immun. 1993 May;61(5):1650–1656. doi: 10.1128/iai.61.5.1650-1656.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tatsumi H., Murakami S., Tsuji R. F., Ishida Y., Murakami K., Masaki A., Kawabe H., Arimura H., Nakano E., Motai H. Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease. Mol Gen Genet. 1991 Aug;228(1-2):97–103. doi: 10.1007/BF00282453. [DOI] [PubMed] [Google Scholar]
  21. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]

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