A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes.
A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6.5 and 7.5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52-76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.
Kang
T H
TH
Department of Biotechnology, Korea University Graduate School, Seoul 136-701, Korea.
Yun
D H
DH
Lee
E H B
EH
Chung
Y B
YB
Bae
Y A
YA
Chung
J Y
JY
Kang
I
I
Kim
J
J
Cho
S Y
SY
Kong
Y
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D001483:Base Sequence; D015153:Blotting, Western; D056646:Cathepsin F; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D045744:Cell Line, Tumor; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D000882:Haplorhini; D015801:Helminth Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D018510:RNA, Helminth / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D000909:Antibodies, Helminth; D015853:Cysteine Proteinase Inhibitors; D015801:Helminth Proteins; D018510:RNA, Helminth; D011994:Recombinant Proteins; D002403:Cathepsins; D056646:Cathepsin F