Title

Isoforms of the Cuticle-degrading Pr1 Protease and Production of a Metalloproteinase by Metarhizium Anisopliae

Document Type

Article

Journal/Book Title/Conference

Archives of Biochemistry and Biophysics

Volume

313

Issue

1

Publication Date

8-1-1994

First Page

1

Last Page

7

DOI

https://doi.org/10.1006/abbi.1994.1350

Abstract

The entomopathogenic fungus, Metarhizium anisopliae, produces three distinct types of proteinases during growth on cockroach cuticle. These were separated by analytical isoelectric focusing and characterized according to their substrate specificity and inhibition patterns as Pr1 subtilisin-like proteinases (four isoforms pI range ∼9.3-10.2), a thermolysin-like metalloproteinase (pI ∼7.3), and trypsin-like serine Pr2 proteinases (two major isoforms, pI ∼4.4 and 4.9 and two minor isoforms, pI ∼5.2). Preparative isoelectric focusing was used to separate the four Pr12 components produced during growth on cockroach cuticle with isoelectric points of 10.2 (m = 30.2 kDa), 9.8 (m = 28.5 kDa), 9.3 (m = 29.5 kDa), and 9.0 (m = 31.5 kDa). Two of the isoforms were also produced, at diminished levels, during growth on elastin or cellulose presumably as a result of carbon and nitrogen derepression. The pI 10.2 Pr1 differed from the other isoforms in preferring alanine over bulky hydrophobic groups at P2 and P3 in discriminating against proline at P2 and in its lack of sensitivity to tetra-butyloxycarbonyl-Gly-Leu-Phe-chlorcomethyl ketone. Differences in the N-terminal amino acid sequences confirmed that the four isoforms are related products of at least two distinct genes. The isoforms showed similar primary specificities, with the aromatic P1 phenylalanine being 10- to 16-fold more reactive than a P1 leucine residue reflected principally in Kcat. However, methionine (containing a long unsubstituted side chain) was also a good substrate for each isoform confirming the low selectivity of their S1 subsites. The isoforms all degraded a variety of solubilized cuticle proteins, with high-molecular-weight acidic proteins being preferentially hydrolyzed. The metalloproteinase is active against the Pr1 substrate succinyl-(Ala)2-Pro-Phe-7-amino-4-coumarin trifluoromethyl, but differs from the Pr1 isoforms in being inhibited by 1,10-phenanthroline and phosphoramidon. The potential role of the metalloproteinase in pathogenicity is discussed.

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