Identification and characterization of a serine protease inhibitor of Clonorchis sinensis.
A gene encoding a serine protease inhibitor of Clonorchis sinensis (CsSERPIN) was identified and characterized. CsSERPIN contained an open reading frame of 1158bp that encoded 385 amino acid residues. Sequence analysis of the primary structure of CsSERPIN revealed that it had essential structural motifs including a reactive central loop (RCL), which well conserved in the serine protease inhibitor (serpin) superfamily. CsSERPIN was classified as a member of the ovalbumin-type serpin family on the basis of phylogenetic analysis and the absence of a classical N-terminal signal peptide. Recombinant CsSERPIN showed an inhibitory effect on chymotrypsin in a dose-dependent manner, but did not effectively inhibit trypsin, thrombin, elastases or cathepsin G. Optimal pH values of CsSERPIN were between 7.0 and 9.0, as evidenced by the rapid loss of inhibitory activity under acidic conditions. CsSERPIN was expressed at various developmental stages of the parasite, from eggs to adult worms, but its expression level was higher in eggs and adult worms than in metacercariae and juvenile worms. CsSERPIN was identified in the soluble extract of the parasite, but not in the excretory and secretory products (ESP) or insoluble extract of the parasite. Immunolocalization analysis of CsSERPIN showed that it mainly localized to the eggs and vitelline glands of the adult worm. These results suggest that intracellular CsSERPIN may be possibly involved in maintaining the physiology of eggs as well as in egg production of C. sinensis by regulating endogenous serine proteases.
Kang
Jung-Mi
JM
Department of Parasitology, Brain Korea 21 Biomedical Center, Gyeongsang National University School of Medicine, Jinju, Republic of Korea.
Sohn
Woon-Mok
WM
Ju
Jung-Won
JW
Kim
Tong-Soo
TS
Na
Byoung-Kuk
BK
D000818:Animals; D003004:Clonorchis sinensis / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D030561:Databases, Nucleic Acid; D004195:Disease Models, Animal; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D016133:Polymerase Chain Reaction; D051381:Rats; D017421:Sequence Analysis; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
D017931:DNA Primers; D018509:DNA, Helminth; D015801:Helminth Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins