Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis

Abstract

Summary Objectives To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen. Methods The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis. Results Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory–secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala–Ala–Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67%, respectively. Conclusions The liver fluke, O. viverrini, has a gut-localized asparaginyl endopeptidase. Refolded recombinant Ov-AEP-1 is catalytically active and has potential for immunodiagnosis of human opisthorchiasis.

Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos

“…Molecular genetic studies of O. viverrini have identified genes encoding various proteins that are stage specific, potential candidates in recombinant form for serodiagnosis (Laha et al 2008, Suttiprapa et al, 2008). For DNA-based methods, a probe targeting repetitive DNA has been used for the detection of eggs (Sirisinha et al, 1991).…”

Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis

“…Recently, the performance of recombinant asparaginyl endopeptidase of O. viverrini was described. However, wide scale deployment of this antigen for serodiagnosis has been hampered by difficulties in refolding the recombinant protease to a natural, soluble conformation [10]. By contrast, the Sm31 antigen or cathepsin B (SmCB-1) of the human blood fluke, Schistosoma mansoni has been widely used for serodiagnosis of schistosomiasis mansoni [11,12].…”

Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: Functional expression, immunolocalization and subsite specificity

“…Soluble extracts from O. viverrini eggs, metacercariae, adult worms, and ES products were prepared as described (Laha et al, 2008). Ten micrograms of each extract and 1.0 μg of recombinant Ov -APR-1 were separated by SDS-PAGE and transferred to nitrocellulose membrane.…”

“…Extensive investigations have been conducted on the phylogeny (Tort et al, 1999; Robinson et al, 2008), structural biology/enzymology (Stack et al, 2008) and vaccinology (Dalton et al, 2003; Hillyer, 2005; McManus and Dalton, 2006) of clan CA proteases from F. hepatica , but substantially less is known about this and other protease families from the human liver flukes (Kang et al, 2004; Kaewpitoon et al, 2008; Laha et al, 2008; Na et al, 2008). …”