title: 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.
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Avanzo
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Jerica
authors.initials: J
authors.lastname: Sabotič
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Sabina
authors.initials: S
authors.lastname: Anžlovar
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Popovič
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Adrijana
authors.initials: A
authors.lastname: Leonardi
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Roger H
authors.initials: RH
authors.lastname: Pain
authors.affiliation: Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia.|Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Janko
authors.initials: J
authors.lastname: Kos
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Jože
authors.initials: J
authors.lastname: Brzin
mesh_terms: D000363:Agaricales / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004331:Drosophila melanogaster; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D005800:Genes, Fungal; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D007306:Insecticides; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors