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

Abstract

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.

• Authors: Thewarach Laha, Jittiyawadee Sripa, Banchob Sripa, Mark Pearson, Leon Tribolet, Sasithorn Kaewkes, Paiboon Sithithaworn, Paul J Brindley, Alex Loukas
• Journal: International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases (Int J Infect Dis) Vol. 12 Issue 6 Pg. e49-59 (Nov 2008) ISSN: 1878-3511 [Electronic] Canada
• PMID: 18619888 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Helminth
• Antigens, Helminth
• Helminth Proteins
• Recombinant Proteins
• Cysteine Endopeptidases
• asparaginylendopeptidase

Topics

• Amino Acid Sequence
• Animals
• Antibodies, Helminth (blood)
• Antigens, Helminth (genetics, immunology, metabolism)
• Cysteine Endopeptidases (genetics, immunology, metabolism)
• Gene Library
• Helminth Proteins (genetics, immunology, metabolism)
• Humans
• Immunoblotting
• Molecular Sequence Data
• Opisthorchiasis (diagnosis, immunology, parasitology)
• Opisthorchis (enzymology, genetics, growth & development, immunology)
• Parasite Egg Count
• Phylogeny
• Predictive Value of Tests
• Recombinant Proteins (genetics, immunology, metabolism)
• Sensitivity and Specificity
• Sequence Analysis, DNA
• Serologic Tests