Isolation, characterization, and cloning of cDNA and the gene for an elastinolytic serine proteinase from Aspergillus flavus

Abstract

An elastinolytic serine proteinase produced byAspergiUusflavus 28 that was isolated from a patient who died of aspergillosis has been purified and characterized. The enzyme was inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The metal-chelating agents EDTA and EGTA [ethylene glycol-bis(1-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not severely inhibit the enzyme. A cDNA and a 2.95-kb segment of genomic DNA containing the proteinase gene were sequenced. The open reading frame that would code for a protein containing 403 amino acids was interrupted by three introns. The mature protein lacks 121 N-terminal amino acids including a putative 21-amino-acid signal peptide. The purified mature protein showed a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas that calculated from the deduced protein sequence was 30 kDa. This elastinolytic serine proteinase ofA. flavus has 83 and 82% sequence homology to the similar proteinases from A. fumigatus and A. oryzae. The catalytic properties and the sequence homology around the putative catalytic amino acids suggest that this enzyme belongs to the serine proteinases of the subtilisin family.

Multiple forms of the serine protease Alp of Aspergillus fumigatus

“…The substrate specificity on synthetic substrates (peptidyl p ‐nitroanilides) was in agreement with the published data on protease Alp . The best‐cleaved substrate, chymotrypsin substrate AAPFpNA, is among the most suitable substrates not only for subtilisin‐like proteases of Aspergilli [ 1, 5, 31–34] but for the enzymes of other fungi as well (e.g. [ 28, 35]).…”

“…[ 7] described five further sequences hybridizing with anti‐ Alp probe at low stringency. Furthermore, in A. flavus [ 18, 34] and A. oryzae [ 39], where subtilisin‐like enzymes homologous to Alp are present, the studies revealed several related sequences. Finally Ramesh and Kolattukudy [ 18] showed evidence for the synthesis of a previously unknown serine protease in A. flavus .…”

“…Protease Alp is rather unstable during purification. Autoproteolysis gives rise to products of molecular weights (SDS‐PAGE) in the range of 18–29 kDa [ 1, 2, 5, 6, 31, 34, 40–42]. At least some of these products retain proteolytic activity (Utz Reichard, personal communication).…”

Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung

“…These results also suggested that the secretion of this enzyme in the host tissue may be targeted to the advancing hyphal tip, supporting the idea that this enzyme probably aids the pathogen to invade the host. Aspergillus flavus, the other major causative agent of aspergillosis, is also known to produce elastinolytic serine protease (18) and elastinolytic metalloprotease (20). The metalloprotease produced byA.…”

Extreme DNA sequence variation in isolates of Aspergillus fumigatus

“…In addition to the sequences listed in Fig. 2, the amino acid sequences from Aspergillus £avus (AFLA, [17]) and Aspergillus niger (ANIG, [10]) were used. Trichoderma harzianum alkaline protease (THAR, [7]) was used as an outgroup in the analysis.…”