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J. Biol. Chem., Vol. 275, Issue 26, 20104-20109, June 30, 2000

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Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis^*

Joe Brzin^§¶, Boris Rogelj^§, Tatjana Popovi^§, Borut trukelj^§, and Anka Ritonja^§

From the ^§ Department of Biochemistry and Molecular Biology, Joef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia and  Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Akereva 7, 1000 Ljubljana, Slovenia

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 M guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K_i = 0.59 nM), cathepsin L (K_i = 0.41 nM), cathepsin B (K_i = 0.48 µM), and bromelain (K_i = 0.16 µM) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).

The amino acid sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P82314.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF230360.

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