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

Abstract

Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other proteases are suspected to be also involved in the degradation of lung structural material. An elastinolytic neutral metalloprotease was purified 320-fold from the extracellular fluid of Aspergilus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molecular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No carbohydrate was attached to this metalloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. Zn2+ and, to a lesser extent, Co2' reversed the inhibition caused by 1,10-phenanthroline. The protease hydrolyzed the peptide bonds His-Leu, Ala-Leu, Tyr-Leu, Gly-Phe, and Phe-Phe in the B chain of insulin. Synthetic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the * Corresponding author. Mailing address:

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases ofCryptococcus neoformans

“…Accordingly, immunogold localization studies demonstrated that A. fumigatus secretes an elastinolytic metalloprotease into invading lung tissue, and Cry. neoformans has been shown to have proteolytic activity against elastin (Muller & Sethi, 1972;Brueske, 1986;Monod et al, 1993;Markaryan et al, 1994). Although the mechanisms that allow Cry.…”

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

“…However, the elution profile by the affinity column and SDS-PAGE of protease indicate that the interaction between bacitracin and protease was specific. Moreover, bacitracin is known in general to interact with proteases in bacitracin affinity chromatography Markaryan et al, 1994;Irvine et al, 1993). Many studies have been published in literature about purification of protease with different methods, as summarized in Table 4.…”

The clinical and pharmacoeconomic analysis of invasive aspergillosis in adult patients with haematological diseases

“…Ground-glass shadowing progressing to diffuse alveolar shadowing was rarely described. 28 The duration of AF therapy in IA is problematic, and there is no clear guidance from published literature. Generally, one should continue treatment until resolution of signs and symptoms, clearance of cultures, improvement of radiological manifestations and improve-ment of the host defences that contributed to the infection.…”