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

1 Department of Biotechnology, Jo ef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia 2 Department of Molecular and Biomedical Sciences, Jo ef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia 3 Faculty of Pharmacy, University of Ljubljana, A ker eva 7, SI-1000 Ljubljana, Slovenia 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. Correspondence Jerica Saboti Jerica.Sabotic{at}ijs.si Abbreviations: B, N -benzoyl; BAPNA, N -benzoyl- DL -arginine- p -nitroanilide; BOC, butoxycarbonyl; CD, circular dichroism; CnSPI, Clitocybe nebularis serine protease inhibitor; (r)Cnp, (recombinant) cnispin; LeSPI, Lentinus edodes serine protease inhibitor; MCA, 7-(4-methyl)coumarylamide; PAA, polyacrylamide; pNA, p -nitroanilide; UTR, untranslated region; Z, benzyloxycarbonyl Present address: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ve na pot 111, SI-1000 Ljubljana, Slovenia. The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are FJ478178 and GQ141891. Supplementary data are available with the online version of this paper.