Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions

Abstract

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K i in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Natural isolates of cnispin also show entomotoxic activity against D. melanogaster, albeit lower than that for cospin (Avanzo et al 2009), validating the entomotoxic potential of the I66 family. Furthermore, cospin showed no toxicity against the nematode C. elegans and amoebozoon A. castellanii.…”

“…A biological role for cospin in the defense of fruiting bodies against Drosophilidae is further corroborated by the absence of trypsin-like protease genes in the C. cinerea genome and by the fact that serine proteases are the predominant digestive proteolytic enzymes in dipterans Terra and Ferreira 2005). In addition to the defensive role directed against exogenous serine proteases, there is some evidence that cnispin and cospin could have a dual role, also participating in the regulation of endogenous serine proteases, in fruiting body development and/or resource recycling (Avanzo et al 2009;). …”

“…This family includes three biochemically characterized members: cospin from C. cinerea , cnispin from C. nebularis (Avanzo Caglič et al 2014;Avanzo et al 2009), and LeSPI (Lentinula edodes serine protease inhibitor) from L. edodes (Odani et al 1999).…”

“…Comparison of expression levels of cnispin in C. nebularis fruiting body and in vegetative mycelium suggests that it is constitutively expressed in mycelium and fruiting body, although expression was higher in the cap of the fruiting body. It was also shown that cnispin is not secreted into the medium (Avanzo et al 2009). …”

“…They inhibit chymotrypsin only weakly and other serine proteases not at all or very weakly. Cospin inhibits trypsin with an equilibrium constant for inhibition (Ki) in the picomolar range and cnispin in the nanomolar range, while both inhibit chymotrypsin with Ki in the micromolar range (Avanzo et al 2009;. The crystal structure of cospin reveals that these proteins are members of the β-trefoil fold protein family.…”