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

Suttiprapa, Sutas, Mulvenna, Jason, Huong, Ngo Thi, Pearson, Mark S., Brindley, Paul J., Laha, Thewarach, Wongkham, Sopit, Sripa, Banchob, and Loukas, Alex (2009) Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: functional expression, immunolocalization and subsite specificity. International Journal of Biochemistry & Cell Biology, 41 (5). pp. 1148-1156.

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Abstract

The human liver fluke Opisthorchis viverrini is endemic in Thailand, Laos and Cambodia where long standing infection is associated with cancer of the bile ducts, cholangiocarcinoma. Here we describe a cathepsin D-like aspartic protease from the gut and other tissues in O. viverrini. Phylogenetic analysis indicated that Ov-APR-1 is cathepsin D-like, conforming with Clan AA, Family A1 of the MEROPS classification. Ov-APR-1 is expressed in the gut of the mature hermaphroditic parasite, in the reproductive tissues including the testis and immature spermatids, and the developing miracidium within the eggshell. The enzyme was also detected in the excretory/secretory products of cultured adult flukes, indicating a role in host-parasite relationships. A recombinant form of the enzyme expressed in Escherichia coli and refolded from denatured inclusion bodies underwent autocatalytic activation and demonstrated hydrolytic activity against the peptide substrate 7-methoxycoumarin-4-acetyl-GKPILFFRLK(DNP)-d-Arg-amide with a kcat/Km = 1.7 × 104 M−1 s−1 and a pH optimum around pH 2.5–3.0. The recombinant enzyme digested hemoglobin and bovine serum albumin. Forty-six serum albumin peptides were detected after digestion with recombinant Ov-APR-1 and sequenced. Like many other aspartic proteases, Ov-APR-1 displayed promiscuous preferences for residues accommodated at the key subsites of the binding pocket although hydrophobic (Leu, Ala, Ile), positively charged (Lys) and bulky aromatic (Phe) residues, in that order, were preferred at P1. Similar residues were accommodated at P1′ although even less selectivity was exerted at this position.

Item ID: 36684
Item Type: Article (Research - C1)
ISSN: 1875-5875
Additional Information:

liver fluke; cholangiocarcinoma; aspartic protease; Cathepsin D; ppisthorchis

Funders: National Institute of Allergy and Infectious Disease (NIAID), Sandler Family Foundation, Thailand Tropical Diseases Research Program (TTDRP) , National Health and Medical Research Council of Australia (NHMRC)
Projects and Grants: NIAID award number UO1AI065871, TTDRP grant number ID02-2-HEL-05-054
Date Deposited: 03 Dec 2014 04:30
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110803 Medical Parasitology @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 100%
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