Macrocypins, a family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera

Abstract

A new family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera has been identified and the family members have been termed macrocypins. These macrocypins are encoded by a family of genes that is divided into five groups with more than 90% within‐group sequence identity and 75–86% between‐group sequence identity. Several differences in the promoter and noncoding sequences suggest regulation of macrocypin expression at different levels. High yields of three different recombinant macrocypins were produced by bacterial expression. The sequence diversity was shown to affect the inhibitory activity of macrocypins, the heterologously expressed macrocypins belonging to different groups showing differences in their inhibitory profiles. Macrocypins are effective inhibitors of papain and cysteine cathepsin endopeptidases, and also inhibit cathepsins B and H, which exhibit both exopeptidase and endopeptidase activities. The cysteine protease legumain is inhibited by macrocypins with the exception of one representative that exhibits, instead, a weak inhibition of serine protease trypsin. Macrocypins exhibit similar basic biochemical characteristics, stability against high temperature and extremes of pH, and inhibitory profiles similar to those of clitocypin from Clitocybe nebularis, the sole representative of the I48 protease inhibitor family in the merops database. This suggests that they belong to the same merops family of cysteine protease inhibitors, the mycocypins, and substantiates the establishment of the I48 protease inhibitor family.

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Even though they have many biochemical properties in common, sequence identity between clitocypin and macrocypin amino acid sequences is low. They are approximately 23 % identical and 33 % similar (Sabotič et al 2007a;Sabotič et al 2006;Sabotič et al 2009). …”

“…Cathepsins B and H, that exhibit both endopeptidase and exopeptidase activity, are not or only very weakly inhibited by mycocypins. In addition to papain-like cysteine proteases (family C1), mycocypins also inhibit asparaginyl endopeptidase (AEP) also called legumain (family C13) while trypsin, but not AEP, is inhibited by macrocypin 4 (Renko et al 2010;Sabotič et al 2007a;Sabotič et al 2009). …”

β-Trefoil inhibitors – from the work of Kunitz onward

“…Recently, reports on the β -trefoil inhibitors of proteases, mainly of plant origin, have been complemented by inhibitors of fungal origin (Brzin et al , 2000 ;Sabotič et al, 2007Sabotič et al, , 2009Renko et al , 2010 ). Currently, there are more than 20 crystal structures of more than 10 different inhibitors with a β -trefoil fold deposited in the PDB (Berman et al , 2000 ).…”

“…Clitocypins and macrocypins were known to inhibit cysteine proteases (Brzin et al , 2000 ;Sabotič et al, 2007Sabotič et al, , 2009 ) but were not classifi ed as inhibitors with the β -trefoil fold until Figure 4 The structure of BASI in complex with subtilisin-like protease savinase. P5-P2 residues in β -strand conformation form numerous hydrogen bonds.…”

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

“…We have previously isolated and characterized a novel family of inhibitors of cysteine proteases from the basidiomycetes Clitocybe nebularis (Brzin et al, 2000;Sabotič et al, 2006Sabotič et al, , 2007a and Macrolepiota procera (Sabotič et al, 2009). Here we describe biochemical properties and evidence for the biological function of new serine protease inhibitors, CnSPIs (Clitocybe nebularis serine protease inhibitors), isolated from C. nebularis, and genetic and biochemical characterization of one of these inhibitors, cnispin (Cnp), which has been heterologously expressed in Escherichia coli.…”