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

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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.

Introduction

The liver fluke, Opisthorchis viverrini, infects more than 6 million people in Thailand alone and the infection has spread into surrounding countries in South-East Asia including Laos and Vietnam (Sripa et al., 2007). People become infected by ingesting raw or fermented fish which form a staple of the diet in rural areas where poor sanitation practices and inadequate sewerage infrastructures prevail. Adult worms reside in the bile ducts, and sequelae of chronic infection can include the hepatobiliary abnormalities cholangitis, hepatomegaly and periportal fibrosis. The most insidious complication of chronic opisthorchiasis, however, is cholangiocarcinoma (CCA), cancer of the bile ducts (Sripa et al., 2007).

The mechanisms by which this liver fluke causes cancer are unknown, but may be multi-factorial. One mechanism by which biliary epithelial cells are thought to become neoplastic is due to excessive and unchecked proliferation stimulated by mitogenic proteins secreted by adult parasites in the biliary tree (Thuwajit et al., 2004, Suttiprapa et al., 2008). To gain a better understanding of the host-parasite interactions underlying molecular pathogenesis of opisthorchiasis, we undertook a shotgun approach to gene discovery for O. viverrini and identified numerous secreted proteins with known roles in other host-parasite systems, including proteases (Laha et al., 2007). Liver fluke proteases, particularly those involved in the digestion of host tissues, are of interest for several reasons including (1) their functional characterization sheds light on their roles in molecular pathogenesis and (2) digestive proteases from other parasitic helminths are efficacious vaccines and drug targets. Indeed, one of the most efficacious vaccine antigens described thus far from the liver fluke of sheep and cattle, Fasciola hepatica, is a cysteine protease expressed in the intestine of the adult fluke (Dalton et al., 2003). Moreover, vaccination of rats with plasmid DNA encoding a cysteine protease from Clonorchis sinensis, a close relative of O. viverrini that infects 30 million people throughout East Asia (Lun et al., 2005), resulted in partial protection against challenge infection (Lee et al., 2006). Cysteine protease inhibitors have shown promise as drugs for fasciolosis (Alcala-Canto et al., 2007) and other parasitic flukes (Abdulla et al., 2007), further highlighting the essential roles these enzymes play in parasite survival.

Almost all of the literature describing proteases from the human liver flukes deals with the C1, and to a lesser extent the C13, families of clan CA cysteine proteases. Extensive investigations have been conducted on the phylogeny (Tort et al., 1999, Robinson et al., 2008), structural biology/enzymology (Stack et al., 2008) and vaccinology (Dalton et al., 2003, Hillyer, 2005, McManus and Dalton, 2006) of clan CA proteases from F. hepatica, but substantially less is known about this and other protease families from the human liver flukes (Kang et al., 2004, Kaewpitoon et al., 2008, Laha et al., in press, Na et al., 2008).

Aspartic proteases belonging to clan AA are less well represented than the cysteine proteases in terms of gene diversity and abundance in parasitic helminths. Their roles have been relatively well characterized in the blood-feeding nematodes (Williamson et al., 2003, Williamson et al., 2004) and trematodes (the schistosomes) (Brindley et al., 2001, Caffrey et al., 2004, Delcroix et al., 2006, Morales et al., 2008), but to our knowledge, there are no reports of aspartic proteases from any species of liver fluke. Here we describe the cloning of a clan AA, family A1 aspartic protease from O. viverrini, its functional expression in E. coli, immunolocalization and detailed characterization of its catalytic activity against blood proteins and its substrate subsite preferences.

Section snippets

Parasites

O. viverrini metacercariae were obtained from naturally infected cyprinoid fish caught by commercial fishermen in fresh water reservoirs in the endemic area of Khon Kaen province, Thailand. The fish were digested by pepsin-HCl. After several washes with normal saline, metacercariae were collected, identified under a dissecting microscope, and viable metacercariae were used to infect hamsters (Mesocricetus auratas) by stomach intubation. The hamsters were maintained at the animal research

An aspartic protease from O. viverrini

The cDNA sequence was 1562 nt in length and encoded a pre-pro-enzyme of 425 amino acids. A signal peptide was present with a cleavage site between Cys-17 and Ser-18 was predicted. Five potential N-glycosylation sites were apparent, two in the pro-region at Asn-45 and -54, and three in the mature protease at Asn-124, -155 and -215. The Ov-apr-1 cDNA sequence has been assigned GenBank accession number DQ131585. The ORF shared 83% identity with the cathepsin D-like sequence from C. sinensis (AF420068

Discussion

Here we report the sequence, phylogeny, tissue localization and substrate specificity of Ov-APR-1 from O. viverrini, the first aspartic protease to be described from any species of liver fluke. Ov-apr-1 mRNA was expressed in all intra-mammalian stages and in the infective stage derived from fish, the metacercaria. The enzyme was localized in the intestine, male and female reproductive organs, and in the miracidium within the developing egg within the uterus of the adult fluke. The widespread

Acknowledgements

This research was supported by award number UO1AI065871 from the National Institute of Allergy and Infectious Diseases (the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID or the NIH), the Sandler Family Foundation, the Thailand-Tropical Diseases Research Program (T-2, grant number ID02-2-HEL-05-054) and the National Health and Medical Research Council, Australia (NHMRC). SS is a Royal Golden Jubilee PhD scholar in the

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