title: Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.
abstract: BACKGROUND
  The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.
  
  
  RESULTS
  As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.
  
  
  CONCLUSIONS
  This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.
authors.affiliation: Center for Advanced Drug Research, Biosciences Division, SRI International, Harrisonburg, Virginia, United States of America.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Watson
authors.forename: Xizhi
authors.initials: X
authors.lastname: Feng
authors.forename: David S
authors.initials: DS
authors.lastname: Askew
authors.forename: Kalyani
authors.initials: K
authors.lastname: Jambunathan
authors.forename: Krishna
authors.initials: K
authors.lastname: Kodukula
authors.forename: Amit K
authors.initials: AK
authors.lastname: Galande
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D001232:Aspergillus fumigatus / Q000201:enzymology*; D016384:Consensus Sequence; D006801:Humans; D007532:Isoleucine; D007930:Leucine; D010447:Peptide Hydrolases / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D019151:Peptide Library; D010649:Phenylalanine; D013045:Species Specificity; D013379:Substrate Specificity; D014443:Tyrosine
chemical_list: D000596:Amino Acids; D019151:Peptide Library; D007532:Isoleucine; D014443:Tyrosine; D010649:Phenylalanine; D010447:Peptide Hydrolases; D007930:Leucine