Prolyl peptidases of the MEROPS S28 family are of particular interest because they are key enzymes in the digestion of proline-rich peptides. A BLAST analysis of the Aspergillus oryzae genome revealed sequences coding for four proteases of the S28 family. Three of these proteases, AoS28A, AoS28B, and AoS28C, were previously characterized as acidic prolyl endopeptidases. The fourth protease, AoS28D, showed high sequence divergence with other S28 proteases and belongs to a phylogenetically distinct cluster together with orthologous proteases from other Aspergillus species. The objective of the present paper was to characterize AoS28D protease in terms of substrate specificity and activity. AoS28D produced by gene overexpression in A. oryzae and in Pichia pastoris was a 70-kDa glycoprotein with a 10-kDa sugar moiety. In contrast with other S28 proteases, AoS28D did not hydrolyze internal Pro-Xaa bonds of several tested peptides. Similarly, to human lysosomal Pro-Xaa carboxypeptidase, AoS28D demonstrated selectivity for cleaving C-terminal Pro-Xaa bonds which are resistant to carboxypeptidases of the S10 family concomitantly secreted by A. oryzae. Therefore, AoS28D could act in synergy with these enzymes during sequential degradation of a peptide from its C-terminus.
Title: Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin Eugster PJ, Salamin K, Grouzmann E, Monod M Ref: Microbiology, 161:2277, 2015 : PubMed
Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.
Dipeptidyl peptidase IV (DPPIV, CD26), a protease-cleaving N-terminal X-Pro dipeptide from selected proteins including some chemokines, is expressed both as a soluble form in plasma and on the cell surface of various immune and nonimmune cell types. To gain insights into the pathophysiological role of CD26 in arthritis, we explored DPPIV/CD26 expression during murine antigen-induced arthritis (AIA), an experimental model of arthritis. AIA induction led to reduced plasma DPPIV activity. In CD26-deficient mice, the severity of AIA was increased as assessed by enhanced technetium uptake and by increased histological parameters of inflammation (synovial thickness and exudate). We demonstrated that CD26 controls the in vivo half-life of the intact active form of the proinflammatory chemokine stromal cell-derived factor-1 (SDF-1). CD26-deficient mice exhibited increased levels of circulating active SDF-1, associated with increased numbers of SDF-1 receptor (CXCR4)-positive cells infiltrating arthritic joints. In a clinical study, plasma levels of DPPIV/CD26 from rheumatoid arthritis patients were significantly decreased when compared to those from osteoarthritis patients and inversely correlate with C-reactive protein levels. In conclusion, decreased circulating CD26 levels in arthritis may influence CD26-mediated regulation of the chemotactic SDF-1/CXCR4 axis.
The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.
A dipeptidyl-peptidase IV was purified from the culture medium of the human-pathogenic fungus Aspergillus fumigatus. The enzyme has an apparent molecular mass of 95 kDa and contained approximately 10 kDa of N-linked carbohydrate. This glycoprotein is antigenic and has all characteristics of the class IV dipeptidyl-peptidases: removal of Xaa-Pro and to a lesser extent Xaa-Ala dipeptides from the N termini of peptides, including bioactive peptides such as neuropeptide Y, [des-Arg1] bradykinin, and glucagon-like peptide 1, activity at neutral pH, and presence in the amino acid sequence of the Gly-X-Ser-X-Gly consensus motif of the serine-hydrolases and the putative catalytic triad (Ser613, Asp690, His725) of the dipeptidyl-peptidases. Moreover, the last 200 amino acids displayed 60 to 65% similarity with the other dipeptidyl-peptidases IV from rat, mouse, human, and yeast. However, unlike the other dipeptidyl-peptidases, the dipeptidyl-peptidase IV of A. fumigatus is a secreted enzyme with a cleavable signal peptide. Expression of a recombinant dipeptidyl-peptidase IV of A. fumigatus has been attained in the yeast Pichia pastoris.
