1887

Abstract

In an acidic protein medium secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in wild-type D141 and in a Δ mutant. Either Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non- fungi.

Funding
This study was supported by the:
  • Swiss National Foundation for Scientific Research (Award 320030-1179641)
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2011-05-01
2021-07-25
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References

  1. Beauvais A., Monod M., Debeaupuis J. P., Diaquin M., Kobayashi H., Latgé J. P. ( 1997a). Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus . J Biol Chem 272:6238–6244 [View Article][PubMed]
    [Google Scholar]
  2. Beauvais A., Monod M., Wyniger J., Debeaupuis J. P., Grouzmann E., Brakch N., Svab J., Hovanessian A. G., Latgé J. P. ( 1997b). Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans. Infect Immun 65:3042–3047[PubMed]
    [Google Scholar]
  3. Beffa T., Staib F., Lott Fischer J., Lyon P. F., Gumowski P., Marfenina O. E., Dunoyer-Geindre S., Georgen F., Roch-Susuki R. et al. ( 1998). Mycological control and surveillance of biological waste and compost. Med Mycol 36:Suppl. 1137–145[PubMed]
    [Google Scholar]
  4. Bergmann A., Hartmann T., Cairns T., Bignell E. M., Krappmann S. ( 2009). A regulator of Aspergillus fumigatus extracellular proteolytic activity is dispensable for virulence. Infect Immun 77:4041–4050 [View Article][PubMed]
    [Google Scholar]
  5. Borg-von Zepelin M., Beggah S., Boggian K., Sanglard D., Monod M. ( 1998). The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol Microbiol 28:543–554 [View Article][PubMed]
    [Google Scholar]
  6. Byun T., Kofod L., Blinkovsky A. ( 2001). Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis. J Agric Food Chem 49:2061–2063 [View Article][PubMed]
    [Google Scholar]
  7. Chevallet M., Luche S., Rabilloud T. ( 2006). Silver staining of proteins in polyacrylamide gels. Nat Protoc 1:1852–1858 [View Article][PubMed]
    [Google Scholar]
  8. Fujinaga M., Cherney M. M., Oyama H., Oda K., James M. N. ( 2004). The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum . Proc Natl Acad Sci U S A 101:3364–3369 [View Article][PubMed]
    [Google Scholar]
  9. Gagnon-Arsenault I., Tremblay J., Bourbonnais Y. ( 2006). Fungal yapsins and cell wall: a unique family of aspartic peptidases for a distinctive cellular function. FEM Yeast Res 6:966–978 [View Article][PubMed]
    [Google Scholar]
  10. Gascuel O. ( 1997). BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol 14:685–695[PubMed] [CrossRef]
    [Google Scholar]
  11. Goloboff P., Farris J., Nixon K. ( 2008). TNT: a free program for phylogenetic analysis. Cladistics 24:774–786 [View Article]
    [Google Scholar]
  12. Guindon S., Gascuel O. ( 2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704 [View Article][PubMed]
    [Google Scholar]
  13. Hall T. A. ( 1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
    [Google Scholar]
  14. Huson D. H., Richter D. C., Rausch C., Dezulian T., Franz M., Rupp R. ( 2007). Dendroscope: an interactive viewer for large phylogenetic trees. BMC Bioinformatics 8:460 [View Article][PubMed]
    [Google Scholar]
  15. Jaton-Ogay K., Paris S., Huerre M., Quadroni M., Falchetto R., Togni G., Latgé J. P., Monod M. ( 1994). Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus . Mol Microbiol 14:917–928 [View Article][PubMed]
    [Google Scholar]
  16. Kämper J. ( 2004). A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis . Mol Genet Genomics 271:103–110 [View Article][PubMed]
    [Google Scholar]
  17. Kataoka Y., Takada K., Oyama H., Tsunemi M., James M. N., Oda K. ( 2005). Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases. FEBS Lett 579:2991–2994 [View Article][PubMed]
    [Google Scholar]
  18. Koaze Y., Goi H., Ezawa K., Yamada Y., Hara T. ( 1964). Fungal proteolytic enzymes. Part I. Isolation of two kinds of acid proteases excreted by Aspergillus niger var macrosporus . Agric Biol Chem 28:216–223 [CrossRef]
    [Google Scholar]
  19. Krappmann S., Bayram O., Braus G. H. ( 2005). Deletion and allelic exchange of the Aspergillus fumigatus veA locus via a novel recyclable marker module. Eukaryot Cell 4:1298–1307 [View Article][PubMed]
    [Google Scholar]
  20. Krappmann S., Sasse C., Braus G. H. ( 2006). Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukaryot Cell 5:212–215 [View Article][PubMed]
    [Google Scholar]
  21. Laemmli U. K. ( 1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [View Article][PubMed]
    [Google Scholar]
  22. Latgé J. P. ( 1999). Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12:310–350[PubMed]
    [Google Scholar]
  23. Léchenne B., Reichard U., Zaugg C., Fratti M., Kunert J., Boulat O., Monod M. ( 2007). Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes. Microbiology 153:905–913 [View Article][PubMed]
    [Google Scholar]
  24. Mattern I. E., van Noort J. M., van den Berg P., Archer D. B., Roberts I. N., van den Hondel C. A. ( 1992). Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Mol Gen Genet 234:332–336 [View Article][PubMed]
    [Google Scholar]
  25. Monod M., Togni G., Rahalison L., Frenk E. ( 1991). Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus . J Med Microbiol 35:23–28 [View Article][PubMed]
    [Google Scholar]
  26. Monod M., Paris S., Sarfati J., Jaton-Ogay K., Ave P., Latgé J. P. ( 1993a). Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus . FEMS Microbiol Lett 106:39–46 [View Article][PubMed]
    [Google Scholar]
  27. Monod M., Paris S., Sanglard D., Jaton-Ogay K., Bille J., Latgé J. P. ( 1993b). Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus . Infect Immun 61:4099–4104[PubMed]
    [Google Scholar]
  28. Monod M., Léchenne B., Jousson O., Grand D., Zaugg C., Stöcklin R., Grouzmann E. ( 2005). Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum . Microbiology 151:145–155 [View Article][PubMed]
    [Google Scholar]
  29. Monod M., Jousson O., Reichard U. ( 2009). Aspergillus fumigatus secreted proteases. Aspergillus fumigatus and Aspergillosis87–106 Latgé J.-P., Steinbach W. J. Washington, DC: American Society for Microbiology; [CrossRef]
    [Google Scholar]
  30. O’Donoghue A. J., Mahon C. S., Goetz D. H., O’Malley J. M., Gallagher D. M., Zhou M., Murray P. G., Craik C. S., Tuohy M. G. ( 2008). Inhibition of a secreted glutamic peptidase prevents growth of the fungus Talaromyces emersonii . J Biol Chem 283:29186–29195 [View Article][PubMed]
    [Google Scholar]
  31. Oda K. ( 2004). Scytalidopepsin B. Handbook of Proteolytic Enzymes, 2nd edn.219–221 Barrett A. J., Rawlings N. D., Woessner J. F. London: Elsevier;
    [Google Scholar]
  32. Punt P. J., Schuren F. H., Lehmbeck J., Christensen T., Hjort C., van den Hondel C. A. ( 2008). Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes. Fungal Genet Biol 45:1591–1599 [View Article][PubMed]
    [Google Scholar]
  33. Reichard U., Büttner S., Eiffert H., Staib F., Rüchel R. ( 1990). Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. J Med Microbiol 33:243–251 [View Article][PubMed]
    [Google Scholar]
  34. Reichard U., Monod M., Rüchel R. ( 1995). Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus . FEMS Microbiol Lett 130:69–74[PubMed]
    [Google Scholar]
  35. Reichard U., Monod M., Odds F., Rüchel R. ( 1997). Virulence of an aspergillopepsin-deficient mutant of Aspergillus fumigatus and evidence for another aspartic proteinase linked to the fungal cell wall. J Med Vet Mycol 35:189–196 [View Article][PubMed]
    [Google Scholar]
  36. Reichard U., Léchenne B., Asif A. R., Streit F., Grouzmann E., Jousson O., Monod M. ( 2006). Sedolisins, a new class of secreted proteases from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase activity at acidic pHs. Appl Environ Microbiol 72:1739–1748 [View Article][PubMed]
    [Google Scholar]
  37. Rolland S., Bruel C., Rascle C., Girard V., Billon-Grand G., Poussereau N. ( 2009). pH controls both transcription and post-translational processing of the protease BcACP1 in the phytopathogenic fungus Botrytis cinerea . Microbiology 155:2097–2105 [View Article][PubMed]
    [Google Scholar]
  38. Ronquist F., Huelsenbeck J. P. ( 2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574 [View Article][PubMed]
    [Google Scholar]
  39. Sasaki H., Tanokura M., Muramatsu T., Nakagawa A., Iwata S., Hamaya T., Takizawa T., Kono T., Takahashi K. ( 1995). X-ray crystallographic study of a non-pepsin-type acid proteinase, Aspergillus niger proteinase A. Adv Exp Med Biol 362:605–609[PubMed]
    [Google Scholar]
  40. Sharon H., Hagag S., Osherov N. ( 2009). Transcription factor PrtT controls expression of multiple secreted proteases in the human pathogenic mold Aspergillus fumigatus . Infect Immun 77:4051–4060 [View Article][PubMed]
    [Google Scholar]
  41. Shevchenko A., Wilm M., Vorm O., Mann M. ( 1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858 [View Article][PubMed]
    [Google Scholar]
  42. Sriranganadane D., Waridel P., Salamin K., Reichard U., Grouzmann E., Neuhaus J. M., Quadroni M., Monod M. ( 2010). Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH. J Proteome Res 9:3511–3519 [View Article][PubMed]
    [Google Scholar]
  43. Takahashi K. ( 2004). Apergillopepsin II. Handbook of Proteolytic Enzymes, 2nd edn.221–224 Barrett A. J., Rawlings N. D., Woessner J. F. London: Elsevier;
    [Google Scholar]
  44. Tilburn J., Scazzocchio C., Taylor G. G., Zabicky-Zissman J. H., Lockington R. A., Davies R. W. ( 1983). Transformation by integration in Aspergillus nidulans . Gene 26:205–221 [View Article][PubMed]
    [Google Scholar]
  45. von Heijne G. ( 1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690 [View Article][PubMed]
    [Google Scholar]
  46. Wilm M., Shevchenko A., Houthaeve T., Breit S., Schweigerer L., Fotsis T., Mann M. ( 1996). Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379:466–469 [View Article][PubMed]
    [Google Scholar]
  47. Yabuki Y., Kubota K., Kojima M., Inoue H., Takahashi K. ( 2004). Identification of a glutamine residue essential for catalytic activity of aspergilloglutamic peptidase by site-directed mutagenesis. FEBS Lett 569:161–164 [View Article][PubMed]
    [Google Scholar]
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