MOLECULAR-CLONING OF THE CDNA AND GENE FOR AN ELASTINOLYTIC ASPARTIC PROTEINASE FROM ASPERGILLUS-FUMIGATUS AND EVIDENCE OF ITS SECRETION BYTHE FUNGUS DURING INVASION OF THE HOST LUNG

Hydrolysis of structural proteins in the lung by extracellular protein ases secreted by Aspergillus fumigatus is thought to play a significan t role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase, We report that A. fumigatus also secretes an aspartic proteinase (asperg illopepsin F) that can catalyze hydrolysis of the major structural pro teins of basement membrane, elastin, collagen, and laminin. The pH opt imum for the enzymatic activity was 5.0 with elastin-Congo red as the substrate, and the activity was not significantly inhibited by pepstat in A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p-nitrophen oxy) propane. The cDNA and gene encoding this aspartic proteinase were cloned and sequenced, The open reading frame, interrupted by three in trons, would encode a protein of 393 amino acids composed of a putativ e 21-amino-acid signal peptide and a 49-amino-acid propeptide precedin g the 323-amino-acid mature protein, The amino acid sequence of A. fum igatus aspartic proteinase has 70, 66, and 67% homology to the sequenc es of those from Aspergillus oryzae, Aspergillus awamori, and Aspergil lus saitoi, respectively. The active-site moth (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a famil y of aspartic proteinases, Polyclonal antibodies were produced in rabb its against both the mature and precursor forms of the aspartic protei nase expressed in Escherichia coli. Immunoblotting with both antibodie s detected a 39-kDa mature enzyme in the culture supernatant of A. fum igatus. The aspartic proteinase activity was inhibited by the antibodi es, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene, Immunogold electron mi croscopy showed that the aspartic proteinase was secreted by A. fumiga tus invading neutropenic mouse lung and its secretion was directed tow ard the germ tubes of penetrating hyphae.