Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding.
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.
Na
B-K
BK
Department of Parasitology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju, Korea. bkna@gnu.ac.kr
Kang
J-M
JM
Cheun
H-I
HI
Cho
S-H
SH
Moon
S-U
SU
Kim
T-S
TS
Sohn
W-M
WM
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
D005353:Fibronectins; D011994:Recombinant Proteins; D003094:Collagen; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L