Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides

Abstract

The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a K i of 5.3 ؋ 10 ؊6 M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a K i of 3 ؋ 10 ؊9 M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion.Many proteinases are synthesized as preproenzymes consisting of a typical signal peptide followed by the propeptide and the mature protein (1). Several functions have been proposed for the propeptide. The propeptide may be required for aiding the proper folding or secretion of the mature proteinase (7,8,28,34), anchoring the proteinase to the membrane and/or maintaining the proteinase in an inactive state until it is released from the cell (33). In several cases, the propeptide plays multiple roles. For instance, the propeptide of a bacterial ␣-lytic proteinase was reported to be involved in the correct folding of mature protein in vivo and in vitro (2, 3, 29-31) and in the inhibition of the activity of the mature proteinase (2). The propeptides of a number of eukaryotic proteinases have been shown to act as strong inhibitors, but their role in folding has not yet been examined. For example, the propeptides of the aspartyl proteinases, pepsin and cathepsin D, inhibit the mature enzymes (11); the propeptide region of the metalloproteinase, carboxypeptidase A, strongly inhibits the mature carboxypeptidase A (27); and a 62-amino-acid pro region of a cysteine proteinase, cathepsin B, strongly inhibits cathepsin B (9). Propeptides of fungal proteinases have not been tested as inhibitors of the respective mature enzymes. During the last several years, extracellular proteinases from different fungal species have been cloned and characterized (10, 13-15, 20, 25, 26, 32, 33). Some of them have large propeptides that in some cases approach the size of the mature enzyme (15,20,32). However, little is known about their function. In this paper, we describe expression of propeptides of a serine pro...

Most of the propeptide is dispensable for stability and autoprocessing of the zymogen of the germination protease of spores of Bacillus species

“…Our new findings indicate that the free propeptide does not inhibit the active enzyme but that two amino-terminal residues on P 41 result in a significant reduction in enzyme activity, and five extra residues result in complete inhibition. These data effectively rule out a model analogous to that proposed for a serine protease and a metalloprotease from Aspergillus fumigatus in which the propeptide not only blocks enzyme activity in the zymogen but even when the propeptide is free (8). Clearly the propeptide residues in GPR must be attached to the protein in order to inhibit the enzyme.…”

Coevolution between pathogen-derived proteinases and proteinase inhibitors of host insects

“…The mature enzyme can be inhibited by its propeptide. 84 A fine-tuned timing of enzyme production has been documented by analysis of gene expression during germination, pathogenesis and conidiogenesis and of the parasitic fungus M. anisopliae. 85 Secretion of thermolysin in order to degrade extracellular matrix proteins and to colonize host tissues occurs in a later stage of mycosis when host hemocytes undergo apoptosis initiated by released destruxins.…”

Functional Characterization of the Propeptide of Plasmodium falciparum Subtilisin-like Protease-1

“…Binding of rp31 and rp31 Mutants to Baculovirus-derived PfSUB-1-The above inhibition data clearly imply tight binding of rp31 and its various derivatives to rPfSUB-1. To directly visualize this, we used a modification of a binding assay described by Kessler and Safrin (29) and Markaryan et al (30). The various recombinant propeptides were subjected to SDS-PAGE and electroblotted onto a membrane.…”