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

Multiple sequence alignment of Ov-AEP-1 with other members of the clan CD, C13 peptidase family. Identical residues in more than 50% of sequences are in black boxes; conserved substitutions are in gray boxes. The conserved active site histidine and cysteine residues are highlighted. The putative N-glycosylation sites are shown on the top of the alignments.

Neighbor-joining phylogenetic tree showing the evolutionary relationship of Ov-AEP-1 with other C13 peptidases from animals and flowering plants. Numbers on branches refer to bootstrap values; the scale bar at the bottom represents 100 changes. The accession numbers of legumain sequences used in the phylogenetic analysis are as follows: ABD64147 (Opisthorchis viverrini), ABQ02437 (Fasciola gigantica), CAC85636 (Fasciola hepatica), P09841 (Schistosoma mansoni), P42665 (Schistosoma japonicum), CAB01126 (Caenorhabditis elegans), CAE75506 (Caenorhabditis briggsae), CAJ45481 (Haemonchus contortus), AAF89679 (Sesamum), AAM60827 (Arabidopsis), CAA04439 (mouse), AAH87708 (rat), CAG33687 (human), AAI11118 (cow), XP_421328 (chicken), AAH56842 (Xenopus), CAG13252 (Tetraodon), and AAS94231 (Ixodes ricinus; tick).

Expression of Ov-aep-1 in developmental stages of O. viverrini, as determined by RT-PCR. The following cDNA templates were included: lane 1, egg; lane 2, metacercaria; lane 3, juvenile worm; lane 4, adult worm; lane 5, adult worm cDNA library. The lower panel shows expression of the actin mRNA in the developmental stages, included here as a constitutively expressed control transcript.

Affinity purification of recombinant Ov-AEP-1 expressed in Escherichia coli as assessed by Coomassie Blue stained SDS-PAGE gel. Lane 1, uninduced E. coli cell pellet; lane 2, IPTG-induced cell pellet; lane 3, supernatant from pellet solubilized in binding buffer; lane 4, supernatant from pellet solubilized in 8M urea in binding buffer; lane 5, affinity-purified Ov-AEP-1 in 6M urea; lane 6, equal loading of refolded purified recombinant Ov-AEP-1 in PBS buffer; lane 7, immunoblot of recombinant Ov-AEP-1 protein probed with anti-hexaHis-tag antibody.

Western blot of recombinant Ov-AEP-1 (rAEP-1), crude somatic (SA), and excretory/secretory (ES) antigens of adult Opisthorchis viverrini probed with purified IgG from the serum of a rabbit immunized with rAEP-1 (α-rAEP-1) or normal rabbit serum (NRS).

Hydrolysis of the fluorogenic peptide Z-Ala–Ala–Asn-aminomethylcoumarin by Opisthorchis viverrini excretory–secretory (ES) products and recombinant Ov-AEP-1 as measured by an increase in relative fluorescence due to release of Z-Ala–Ala–Asn-aminomethylcoumarin over time. The assay was performed at pH 7. 0. Inclusion of the inhibitor iodoacetamide at 5mM completely inhibited the activity of Ov-AEP-1 (not shown).

Immunohistochemical localization of legumain in Opisthorchis viverrini adult worms. Anti-Ov-AEP-1 rabbit IgG bound strongly to the gut (panel A) and eggs in the uterus (panel B). Control IgG from the same rabbit prior to immunization did not bind to the same structures (panels C and D). Scale bar are shown.

Immunoblot analysis of recognition of recombinant Ov-AEP-1 by sera from people with parasitologically proven infection with Opisthorchis viverrini (A). ‘+’ and ‘−’ indicate positive or negative scores, respectively, for the presence of a band at 47kDa representing recognition of the recombinant antigen. C represents sera pooled from several uninfected control donors. Sera from patients with other parasitic infections did not recognize recombinant Ov-AEP-1 (B). H=hookworm infection; E=echinostomiasis; T=Taenia infection; M=minute intestinal fluke infection.