期刊
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
卷 41, 期 5, 页码 1148-1156出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.biocel.2008.10.013
关键词
Liver fluke; Cholangiocarcinoma; Aspartic protease; Cathepsin D; Opisthorchis
资金
- National Institute of Allergy and Infectious Diseases [UO1AI065871]
- Sandler Family Foundation
- Thailand-Tropical Diseases Research Program [ID02-2-HEL-05-054]
- National Health and Medical Research Council, Australia (NHMRC)
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 call and refolded from denatured inclusion bodies underwent autocatalytic activation and demonstrated hydrolytic activity against the peptide substrate 7-methoxycoumarin-4-acetyl-GKPILFFRLK(DNP)-D-Argamide 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. (C) 2008 Elsevier Ltd. All rights reserved.
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