title: Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: functional expression, immunolocalization and subsite specificity.
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 k(cat)/K(m)=1.7 x 10(4)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.
authors.affiliation: Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Sutas
authors.initials: S
authors.lastname: Suttiprapa
authors.forename: Jason
authors.initials: J
authors.lastname: Mulvenna
authors.forename: Ngo Thi
authors.initials: NT
authors.lastname: Huong
authors.forename: Mark S
authors.initials: MS
authors.lastname: Pearson
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
authors.forename: Thewarach
authors.initials: T
authors.lastname: Laha
authors.forename: Sopit
authors.initials: S
authors.lastname: Wongkham
authors.forename: Sasithorn
authors.initials: S
authors.lastname: Kaewkes
authors.forename: Banchob
authors.initials: B
authors.lastname: Sripa
authors.forename: Alex
authors.initials: A
authors.lastname: Loukas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002402:Cathepsin D / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D003530:Cyprinidae / Q000469:parasitology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009891:Opisthorchis / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D