Sedolisins, a New Class of Secreted Proteases fromAspergillus fumigatuswith Endoprotease or Tripeptidyl-Peptidase Activity at Acidic pHs

Abstract

The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17-to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide-p-nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-HisPro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin

“…Large antigen peptides (350-400 aa) corresponding to partial amino acid sequences of A. oryzae and Aspergillus flavus putative proteases of the S28 family were produced using plasmid pET-11aH6, a derivative of pET-11a made for His 6 -tagged peptide production (Reichard et al, 2006). For the synthesis of peptides corresponding to partial amino acid sequences of three A. oryzae proteases (S28B, S28C and S28D; see Results), sense and antisense primer pairs P3/P4, P5/P6 and P7/P8 (Table 2) were used to amplify A. oryzae RIB40 (NBRC 100959) genomic DNA.…”

“…At optimal pH, AoS28A (AoS28B) activity was 2 (37), 16 (240) and 33 (38) mmol min 21 mg 21 , respectively. Both enzymes showed no activity on the DppIV substrate AP-pNA (Doumas et al, 1998) and on the sedolisin substrate APF-pNA (Reichard et al, 2006).…”

“…Exoproteases secreted at neutral or alkaline pH are leucine aminopeptidases of the M28 family and an Xprolyl exopeptidase (DppIV) of the S9 family (see MEROPS peptidase database; http://merops.sanger.ac.uk/) (Beauvais et al, 1997;Blinkovsky et al, 2000;Chien et al, 2002;Doumas et al, 1998;Monod et al, 2009). Exoproteases secreted at acidic pH are tripeptidyl peptidases of the sedolisin family and prolyl endopeptidases of the S28 family (Reichard et al, 2006;Sriranganadane et al, 2010). Leucine aminopeptidases and tripeptidyl peptidases are non-specific amino peptidases, but are not able to cut before and after a proline residue (Byun et al, 2001;Sriranganadane et al, 2010).…”

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium

“…fumigatus gprAD mutants were identified by PCR of genomic DNA from various numbers of the hygromycin-resistant colonies as previously performed for selection of other protease-and transporter-deficient mutants (Reichard et al, 2006;Léchenne et al, 2007). Two pairs of specific DgprA primers (P5/P6 and P7/P8, Table 1) were used to verify correct gprA replacement.…”

“…In a protein medium at low pH, A. fumigatus was found to secrete another set of proteases, including the aspartic protease Pep as an endoprotease (Reichard et al, 1995), tripeptidyl-peptidases (Tpp) of the sedolisin family (SedB to SedD) (Reichard et al, 2006), and AfuS28 and carboxypeptidases of the S10 family as exoproteases (Sriranganadane et al, 2010). In addition, we identified by MS an as yet uncharacterized putative glutamic endoprotease, AfuGprA (XP_748619, MER107323; encoding a gene at the locus AFUA_3G02970) (Monod et al, 2009;Sriranganadane et al, 2010), homologous to Aspergillus niger proteinase A or aspergillopepsin II (Takahashi, 2004) and to Scytalidium lignicolum scytalidopepsin B (Oda, 2004).…”