Comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors
Section snippets
Materials and methods
Enzymes. Human cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) were purified as described [12]. 2× crystallized papain (EC 3.4.22.2) was further purified by affinity chromatography [13]. Recombinant human cathepsin K [14] and caspases 3, 6 and 7 [15] all expressed in E. coli and legumain isolated from pig kidney cortex [16] were provided by prof. Boris Turk (our laboratory).
Isolation of natural clitocypin. Basidiocarps or fruiting bodies of C. nebularis (also classified as Lepista
Purification and properties of natural clitocypin (nClt)
Approximately 20 mg of purified clitocypin was obtained from 1 kg of fresh basidiocarps. On SDS–PAGE under denaturing conditions it gave a band of 17 kDa (Fig. 5A, lane 5) and, on IEF, a single band with pK 4.7 (Fig. 5B, lane 1). The protein gave a single mass of 16,863 Da by ESI mass spectrometry. 8-cycle sequencing of purified nClt revealed a single N-terminus, H2N-LEDGIYRL, but with rather low sequence yield. The recent finding provided by the cDNA derived sequences [11] showed that the
Discussion
Our long-term goal is to determine the biochemical properties, mode of interaction and crystallographic structure of clitocypin, the sole member of the mycocypins characterized from C. nebularis [9] but recently detected also in some other mushroom species (J. Brzin and J. Sabotič, unpublished results). We have shown that the natural clitocypin comprises a mixture of closely related isoforms [11]. The aim of this work was to express and purify recombinant clitocypin and compare its properties
Acknowledgments
This work was supported by the Slovenian Research Agency Grant No. P4-0127. The authors acknowledge helpful discussion of the results and critical reading of the manuscript to Dr. Roger Pain. We are grateful to Dr. Boris Turk for kindly providing the cathepsin K, legumain and caspases, and to Dr. Bogdan Kralj for performing ESI mass spectrometry measurements.
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Structural and functional studies of legumain–mycocypin complexes revealed a competitive, exosite-regulated mode of interaction
2022, Journal of Biological ChemistryClitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases
2015, Pesticide Biochemistry and PhysiologyCitation Excerpt :Macrocypins from parasol mushroom constitute family I85 in the MEROPS classification and belong to the mycocypin superfamily of cysteine protease inhibitors unique to basidiomycetes [22,23]. Clitocypin (Clt) is a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis) that belongs to family I48 of the MEROPS classification [24–27] and, together with macrocypins, forms the mycocypin superfamily. They possess the β-trefoil fold that affords them resistance to proteolytic degradation and tolerance to extreme pH and temperatures [28,29].
Proteins of higher fungi - from forest to application
2012, Trends in BiotechnologyCitation Excerpt :Information on the latter is even more limited [16]. The only two well-characterized families of cysteine protease inhibitors possess characteristics not present in structurally similar protease inhibitors from plant or animal sources [6,9]. Two types of serine protease inhibitor have been characterized; those homologous to propeptides of subtilisin-like proteases from the oyster mushroom Pleurotus ostreatus [17], and a family of trypsin-specific inhibitors with a β-trefoil fold [18,19].
Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies
2011, BiochimieCitation Excerpt :Both clitocypin and macrocypins have a β-trefoil fold formed by a core six-stranded β-barrel which supports 11 loops that provide a versatile surface for inhibition of several types of proteases [4]. The inhibitory profile of mycocypins is broad, including many papain-like cysteine proteases (family C1, clan CA), legumain (family C13, clan CD) and the serine protease trypsin (family S1, clan PA) [1,2,5]. Furthermore, mycocypins are small and exceptionally stable proteins, exhibiting high thermal and broad pH stability [2,6].