Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis

A gene encoding a homologue of phospholipase A2 was identified from the Clonorchis sinensis adult cDNA plasmid library. The deduced amino acid sequence including a signal peptide that has 28–46% identity with secretory phospholipase A2, group III (group III sPLA2) of other species. It also has typical features of group III sPLA2s including 10 cysteines, the key residues of the Ca2+ loop and catalytic site. The recombinant protein encoded by this gene expressed in Escherichia coli showed a product of about 34 kDa in SDS-PAGE. Prediction of signal peptide and Western blot analysis indicated the group III secretory phospholipase A2 of C. sinensis (CsGIIIsPLA2) was an excretory–secretory product (ES product). The enzyme activity of the recombinant protein was determined using phosphatidylcholine as substrates. The result revealed that the protein was a Ca2+-dependent PLA2. Both MTT test and cell cycle analysis of LX-2 showed a higher percentage of cells are in proliferation phase. Semi-quantitative RT-PCR experiments demonstrated an up-regulated expression of collagen III in these cells after incubation with the recombinant protein. We also identified that the recombinant CsGIIIsPLA2 could bind to some membrane proteins on LX-2 cells specifically by immunofluorescence, thus there might be receptors of CsGIIIsPLA2 on the LX-2 cell membrane. Our results suggest that CsGIIIsPLA2 might play an important role in the initiation and development of hepatic fibrosis caused by C. sinensis.