PURIFICATION AND CHARACTERIZATION OF AN ELASTINOLYTIC METALLOPROTEASEFROM ASPERGILLUS-FUMIGATUS AND IMMUNOELECTRON MICROSCOPIC EVIDENCE OFSECRETION OF THIS ENZYME BY THE FUNGUS INVADING THE MURINE LUNG

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 Aspergillus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molec ular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyac rylamide gel electrophoresis. No carbohydrate was attached to this met alloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely i nhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by i nhibitors 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. Synthet ic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the fluorimetri c assay of the A. fumigatus metalloprotease. This enzyme had maximum a ctivity in the pH range 7.5 to 8.0 and at 60 degrees C. It retained 50 % of the protease activity when held at 60 degrees C for 1 h. Zn2+ and Co2+ at 1 mM did not inhibit the protease activity. The metalloprotea se was able to hydrolyze elastin, and its elastinolytic activity was c omparable to that of the serine protease from this organism. The prese nce of Zn2+ in the culture medium stimulated the metalloprotease produ ction. Rabbit antibodies prepared against the enzyme severely inhibite d the enzyme activity. Immunogold electron microscopy revealed that A. fumigatus invading neutropenic mouse lungs secretes this metalloprote ase.