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Searching the secretome of Opisthorchis viverrini for growth factor-like molecules

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.