Open AccessStructural and Immunological Characteristics of a 28-Kilodalton Cruzipain-Like Cysteine Protease ofParagonimus westermaniExpressed in the Definitive Host Stage
Author(s) -
Doo-Hee Yun,
Jinwook Chung,
YongAn Chung,
Young-Yil Bahk,
Sung-Soo Kang,
Yoon Kong,
Seung Yull Cho
Publication year - 2000
Publication title -
clinical and diagnostic laboratory immunology
Language(s) - English
Resource type - Journals
eISSN - 1098-6588
pISSN - 1071-412X
DOI - 10.1128/cdli.7.6.932-939.2000
Subject(s) - paragonimus westermani , biology , complementary dna , proteases , cysteine protease , cysteine , microbiology and biotechnology , peptide sequence , biochemistry , antigenicity , signal peptide , amino acid , cathepsin c , open reading frame , recombinant dna , protease , enzyme , antigen , gene , paragonimiasis , genetics , zoology , helminths
A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.