Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis.
OBJECTIVE
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.
Laha
Thewarach
T
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. thewa_la@kku.ac.th
Sripa
Jittiyawadee
J
Sripa
Banchob
B
Pearson
Mark
M
Tribolet
Leon
L
Kaewkes
Sasithorn
S
Sithithaworn
Paiboon
P
Brindley
Paul J
PJ
Loukas
Alex
A
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood*; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015723:Gene Library; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009889:Opisthorchiasis / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D009891:Opisthorchis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D010270:Parasite Egg Count; D010802:Phylogeny; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012698:Serologic Tests; D000099240:Asparaginyl Endopeptidase
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D003546:Cysteine Endopeptidases; D015801:Helminth Proteins; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase