title: Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding.
abstract: Cryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.
authors.affiliation: Department of Parasitology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju, Korea. bkna@gnu.ac.kr
authors.forename: B-K
authors.initials: BK
authors.lastname: Na
authors.forename: J-M
authors.initials: JM
authors.lastname: Kang
authors.forename: H-I
authors.initials: HI
authors.lastname: Cheun
authors.forename: S-H
authors.initials: SH
authors.lastname: Cho
authors.forename: S-U
authors.initials: SU
authors.lastname: Moon
authors.forename: T-S
authors.initials: TS
authors.lastname: Kim
authors.forename: W-M
authors.initials: WM
authors.lastname: Sohn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D016785:Cryptosporidium parvum / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D034101:Sporozoites / Q000378:metabolism
chemical_list: D005353:Fibronectins; D011994:Recombinant Proteins; D003094:Collagen; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L