The International Journal of Biochemistry & Cell Biology
Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: Functional expression, immunolocalization and subsite specificity
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
References (44)
- et al.
Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation
J Biol Chem
(1995) - et al.
Proteolysis of human hemoglobin by schistosome cathepsin D
Mol Biochem Parasitol
(2001) - et al.
Blood ‘n’ guts: an update on schistosome digestive peptidases
Trends Parasitol
(2004) - et al.
Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines
Int J Parasitol
(2003) - et al.
A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite
J Biol Chem
(2006) - et al.
Cathepsin B-like cysteine proteases and Caenorhabditis elegans homologues dominate gene products expressed in adult Haemonchus contortus intestine
Mol Biochem Parasitol
(2001) - et al.
Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease
Exp Parasitol
(2004) - et al.
Vaccination with DNA encoding cysteine proteinase confers protective immune response to rats infected with Clonorchis sinensis
Vaccine
(2006) - et al.
Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus
Mol Biochem Parasitol
(1997) - et al.
Clonorchiasis: a key foodborne zoonosis in China
Lancet Infect Dis
(2005)
RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade
Mol Biochem Parasitol
CsCF-6, a novel cathepsin F-like cysteine protease for nutrient uptake of Clonorchis sinensis
Int J Parasitol
A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus
Mol Biochem Parasitol
Proteomic and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen, Fasciola hepatica: expansion of a repertoire of virulence-associated factors
Mol Cell Proteomics
Comparison of adult somatic and cysteine proteinase antigens of Fasciola gigantica in enzyme linked immunosorbent assay for serodiagnosis of human fasciolosis
Acta Trop
Engineering of porcine pepsin. Alteration of S1 substrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis
J Biol Chem
Localisation of parasite antigens and inflammatory responses in experimental opisthorchiasis
Int J Parasitol
Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica
J Biol Chem
Origin of hemoglobin in the cecal contents of Fasciola hepatica
Exp Parasitol
Proteinases and associated genes of parasitic helminths
Adv Parasitol
Digestive proteases of blood-feeding nematodes
Trends Parasitol
A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms
J Biol Chem
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