Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A(2) and investigation of its potential contribution to hepatic fibrosis.
A gene encoding a homologue of phospholipase A(2) 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 A(2), group III (group III sPLA(2)) of other species. It also has typical features of group III sPLA(2)s including 10 cysteines, the key residues of the Ca(2+) loop and catalytic site. The recombinant protein encoded by this gene expressed in Escherichia coli showed a product of about 34kDa in SDS-PAGE. Prediction of signal peptide and Western blot analysis indicated the group III secretory phospholipase A(2) of C. sinensis (CsGIIIsPLA(2)) 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 Ca(2+)-dependent PLA(2). 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 CsGIIIsPLA(2) could bind to some membrane proteins on LX-2 cells specifically by immunofluorescence, thus there might be receptors of CsGIIIsPLA(2) on the LX-2 cell membrane. Our results suggest that CsGIIIsPLA(2) might play an important role in the initiation and development of hepatic fibrosis caused by C. sinensis.
Hu
Fengyu
F
Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China.
Hu
Xuchu
X
Ma
Changling
C
Zhao
Junhong
J
Xu
Jin
J
Yu
Xinbing
X
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000472:pathogenicity*; D003067:Coenzymes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008103:Liver Cirrhosis / Q000469:parasitology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism; D054497:Phospholipases A2, Secretory / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*
D003067:Coenzymes; D015801:Helminth Proteins; D010713:Phosphatidylcholines; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D037521:Virulence Factors; D054497:Phospholipases A2, Secretory; D002118:Calcium