Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli

Abstract

An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis patient/and an internal peptide derived from it were subjected to N-terminal sequencing. Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme. The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame. According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase. This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide. A. fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases. The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme. Although the

Virulence factors of medically important fungi

“…A. fumigatus possesses two elastases, one a serine protease and the other a metalloproteinase, both of which have been cloned and sequenced (143,193,200,202,203,266,269,280,281). The serine protease appears to be identical to a previously described 32-kDa alkaline protease (143,200,202,203,269,280,281).…”

“…Antibodies had no effect in an immunosuppressed-mouse model but protected immunocompetent mice given a massive inoculum of conidia. Compared with the serine protease, the role of the A. fumigatus metalloproteinase as a putative virulence factor has not been exten-sively studied (193,266). Immunogold electron microscopy revealed that A. fumigatus organisms invading neutropenic mouse lungs secrete this metalloprotease (193).…”

Characterization of the Aspergillus nidulans aspnd1 gene demonstrates that the ASPND1 antigen, which it encodes, and several Aspergillus fumigatus immunodominant antigens belong to the same family

“…These locations are in agreement with those described for certain genes from other Aspergillus spp. (5,38).…”

Multiple forms of the serine protease Alp of Aspergillus fumigatus

“…However, in the protease‐deficient mutants created by disruption of the Alp gene (‘gene knockout’) no alteration of virulence in comparison to the wild‐type strains and revertants could be found [ 8, 9]. Later on an extracellular zinc‐containing metalloprotease Mep was isolated [ 10, 11] and the structure of the respective gene described [ 12, 13]. However, in the double disruptant alp‐mep‐ (that was devoid of proteolytic activity on standard media) no decrease in virulence was found [ 12].…”