Cryptopain-1, a cysteine protease of Cryptosporidium parvum, dose not require prodomain for folding

초록

Cryptosporidium parvum is an intracellular protozoan parasite which causes cryptosporidiosis in mammals including human. In this study, we identified a gene encoding cysteine protease of C. parvum (cryptopain-1) and characterized biochemical properties of the recombinant protein. Multiple sequence alignment of the deduced amino acid sequence of cryptopain-1 with those of previously reported cysteine proteases from other protozoan parasites showed that cryptopain-1 shares common structural properties including the presence of characteristic prodomain and conservation of active site amino acid residues for cathepsin L-like cysteine proteases. A construct harboring a portion of the prodomain and the mature domain of cryptopain-1 was expressed in Escherichia coli and refolded to active enzyme. It showed typical biochemical properties of cathepsin L-like cysteine proteases including pH optimum at acidic pH and requirement of reducing condition for activity. To better characterize the determinants of folding for cryptopain-1, multiple constructs with or without different length of prodomain of the enzyme were expressed in E. coli and assessed their abilities to refold. All the constructs except a construct that did not contain full-length mature domain were successfully refolded into active enzymes. It suggests that cryptopain-1 dose not require prodomain for folding. Cryptopain-1 preferentially degraded fibrillar proteins including collagen and fibronectin, but not globular proteins. This suggests its possible role for host cell invasion and/or egress of the parasite.