Aspergillus Protein Degradation Pathways with Different Secreted Protease Sets at Neutral and Acidic pH

Abstract

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

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

“…A 1059 bp DNA fragment encoding part (aa 175-527) of a fourth putative protease of the S28 family in A. flavus (MER 161166) but not identified in the A. oryzae genome was synthesized by Genecust. This protein, called S28A, was 64 % identical to the previously characterized AfuS28 (Sriranganadane et al, 2010) and appeared to be a putative orthologue of this prolyl endopeptidase. The PCR products and synthesized DNA were digested with either Nco I or Rca I and Bam HI to be subsequently cloned into the Nco I and Bam HI sites of pET-11aH6.…”

“…Culture supernatant was concentrated by ultrafiltration and desalted as described above. Thereafter, His 6 -tagged AoS28A was extracted with a Ni-NTA Agarose (Life Technologies) resin column with histidine elution buffer (50 mM histidine in PBS) as described previously (Sarfati et al, 2006;Sriranganadane et al, 2010). For AoS28B purification, the active desalted fractions eluted from the PD10 column were pooled and applied to an HPT (Bio-Rad) column that had been previously equilibrated with a 10 mM sodium phosphate buffer (pH 7.0).…”

“…These fungi produce different sets of endo-and exoproteases that degrade extracellular protein sources into amino acids, and di-and tripeptides that are assimilable via transporters (Monod et al, 2009;Sriranganadane et al, 2010). During this process, the main function of endoproteases is to produce a large number of free ends on which exoproteases may act.…”

“…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).…”

“…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). Conversely, X-Pro sequences can be removed at neutral pH by DppIV and X-X-Pro sequences are trimmed off at acidic pH by prolyl endopeptidases of the S28 family.…”