Structural and immunological characteristics of a 28-kilodalton cruzipain-like cysteine protease of Paragonimus westermani expressed in the definitive host stage

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adul t cDNA library, The cDNA contained a single open reading frame of 975 bp en coding 325 amino acids, which exhibited the structural motif and domain org anization characteristic of cysteine proteases of non-cathepsin Bs includin g a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine re sidues as well as active sites in the mature catalytic region, Analysis of its phylogenetic position revealed that this novel enzyme belonged to the c ruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determi ned from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a lo cation in the intestinal epithelium, suggesting that this enzyme could be s ecreted and involved in nutrient uptake and immune modulation. The recombin ant protein expressed in Escherichia coli was used to assess antigenicity b y immunoblotting with sera from patients with active paragonimiasis and fro m 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.