Structural and Immunological Characteristics of a 28-Kilodalton Cruzipain-Like Cysteine Protease ofParagonimus westermaniExpressed in the Definitive Host Stage

Abstract

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.Paragonimus westermani is a trematode parasite that causes chronic inflammatory lung disease as well as systemic infection including cerebral invasion in humans and carnivorous mammals. Human infection occurs by ingesting undercooked freshwater crayfish or crabs containing metacercaria or eating raw boar meat. The metacercariae excyst in the duodenum, penetrate the peritoneal cavity, and finally, migrate to the lung, in which they become adults and are surrounded by a thick granulomatous wall (1, 18). The adult worms can survive for approximately 5 years.Parasite cysteine proteases are known to play critical roles in parasitic infections. They participate in a broad range of biological processes including egg hatching and subsequent stage transitions, invasion and migration through host tissues, and immune modulation and nutrient uptake (4,25,28,29,39,43). Recent studies have shown that these enzymes might be good targets for the development of vaccines (20, 33) and antiparasitic drugs (7, 9).In P. westermani, at least six different species of cysteine protease with molecular sizes of 53, 34, 28, 27, 17, and 15 kDa, respectively, have been characterized in eggs, metacercariae, juveniles, and adults (4,5,14,22,45). The 28-and 27-kDa proteases released by the metacercariae have been shown to share biochemical features with cathepsin L of F. hepatica (8) or with cathepsin S of sparganum (25) and are believed to play important roles in metacercarial excystment, tissue migration, and immune evasion (4, 6, 14, 45).It is of particular interest that P. westermani possesses multiple cysteine proteases of similar sizes ranging from 29 to 27 kDa (4...

Molecular and biochemical characterization of hemoglobinase, a cysteine proteinase, in Paragonimus westermani

“…As their various functions, cysteine proteinases of P. westermani have been reported to localize in various tissues of the worm, such as, the epithelia and luminal contents of the intestine (Yang et al, 2004), vitelline glands , intestinal epithelium and parenchymal cells (Yun et al, 2000). In our immunohistochemical analysis of the hemoglobinase using polyclonal antibodies against recombinant protein, a positive signal was observed in the intestinal epithelium of the adult, which concurs with its expected pH optimum.…”

Opisthorchis viverrini: The carcinogenic human liver fluke

“…This is even higher than conventional indirect ELISA using O viverrini somatic extracts or excretory-secretory product antigen [63,64,79,81] . The sensitivity and specificity are similar to other studies using ELISA based on recombinant trematode cysteine protease, such as C sinensis (sensitivity: 81.3%-96%; specificity: 92.6%-96.2%) [84,85] and P. westermani (sensitivity: 86.2%-93%; specificity: 93%-98%) [86,87] . It seems that the more specific purified antigen used, the more sensitivity and specificity obtained.…”

Evaluation of Clonorchis sinensis Recombinant 7-Kilodalton Antigen for Serodiagnosis of Clonorchiasis

“…A number of antigens of molecular masses ranging 8 to 90 kDa (1, 12) prepared differently from either crude worm extracts or ESP have been reported to react strongly to human clonorchiasis. However, some cross-reactions against serum samples obtained from patients with other trematode infections, such as paragonimiasis, fascioliasis, metagonimiasis and schistosomiasis japonica, were observed (1,7,8,13). It seemed difficult to minimize serological cross-reactivity between some trematode infections in previous studies.…”