| Epidemiological studies have shown that the cytotoxin-associated protein A (CagA) secreted by H.pylori is closely associated with chronic active gastrics, peptic ulcers and gastric cancer. CagA is encoded by the cag pathogenicity island (cag PAI) and is translocated into host cells through a type IV secretion system. Tyrosine-phosphorylation of EPIYA repeat sequences located in C-terminal region is considered playing an important role in the biological function of CagA. By specifically binding and activating SHP-2 phosphatase, the phosphorylated CagA disturbs signaling pathway and thereby results in cell proliferation and differentiation. The activation of SHP-2 also induces the rearrangement of cytoskeletal structure in host cells, enhancing their ability of motility and migration. However, the pathogenesis of CagA remains unclear. Proteomic analysis of host cells affected by CagA will provide full understanding of host response to infection at the protein level. The ProteinChip platform, based on surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) can be used to establish protein profiles under different physiologic and pathologic conditions. This innovative proteomic technology has certain advantages over other methods such as two-dimensional gel electrophoresis (2-DE). SELDI has higher throughput capability, subfemtomole range sensitivity and higher resolution at low mass ranges. It has been successfully used in the biomarker discovery as well as early detection of cancer and has a bright prospect of application in basic research. In the present study, the proteome of AGS cells transfected with cagA gene was analyzed using SELDI-TOF-MS to identify the host cell proteins involved in CagA induced pathogenesis and further investigate whether the changes of protein expressiondepend on the tyrosine-phosphorylation of CagA as well as the relationship with the activation of Erk/MAPK signaling pathway. We hope to discover disease related proteins with low mass range and/or low abundance, which is difficult to be detected by traditional techniques.MethodThree groups of AGS cells were transfected with wild type cagA, phosphorylation site mutant cagA and pcDNA3.1(+) plasmid respectively. Another group transfected with EGFP/ pcDNA3.1(+) was used to evaluate the transfection rate. RT-PCR and sequencing were employed to confirm the expression of wild type and mutant cagA in the target cells. Protein expression profile of each group was then analyzed by ProteinChips and SELDI-TOF-MS. For each detected protein ProteinChip software version 3.1 runs the Mann-Whitney statistical test (the nonparametric equivalent of the analysis of variance) to assess the significance of expression differences among different sample groups. To investigate whether the expression differences are caused by the activation of Erk/MAPK signaling pathway, another six groups respectively treated with Erkl/2 inhibitor or DMSO after transfection were also analyzed by ProteinChips and SELDI-TOF-MS. Western-blot was employed to analyze the level of phosphorylated Erkl/2.Result1. Approximate 200 proteins with mass range of 15OO~3OOOO Da (signal-to-noise ratio>5.0 and relative-peak-intensity >1.0) were obtained using SAX2 or CM10 ProteinChip arrays respectively.2. Sixteen proteins were found with significant expression difference between wild type cagA transfected AGS cells and vector transfected cells (P<0.05). Ten proteins with m/z of 4.2kD^ 4.71 kD, 4.73 kD, 5.1 kD, 6.5 kD, 6.7 kD> 8.2 kD, 9.1 kD, 13.8 kD, 14.0 kD were up-regulated while 6 proteins with m/z of 2.0 kD, 4.3 kD, 8.6 kD, 10.0 kD, 11.1 kD, 11.6 kD were down-regulated in the wild type cagA transfected AGS cells.3. Only 4 proteins at m/z of 4.71 kD, 4.73 kD, 6.5 kD and 6.7 kD among the 16 proteins mentioned above were found up-regulated after mutant cagA transfection. The expression level of other 12 proteins in the mutant cagA transfected cells was found have no significant difference with vector transfected cells.4. The expression level of three proteins at m/z of 4.2 kD, 8.2 kD> 9.1 kD were found up-regulated in the wild type cagA transfected cells in which Erkl/2 was activated. When treated with Erkl/2 inhibitor after transfection, the peak values of these 3 proteins were decreased to the level of cells with vector transfection. The expression differences of other 13 proteins remained either in Erk active or inactive condition.Conclusion1. Sixteen host proteins at m/z of 2.0 kD, 4.2 kD, 4.3 kD, 4.71 kD. 4.73 kD, 5.1 kD, 6.5kD, 6.7kD, 8.2kD, 8.6kD, 9.1 kD, lO.OkD, 11.1 kD, 11.6kD, 13.8 kD, 14.0 kD are associated with the pathogenesis of CagA.2. The changes of 12 proteins' expression at m/z of 2.0 kD, 4.2 kD, 4.3 kD, 5.1 kD, 8.2 kD, 8.6 kD, 9.1 kD, 10.0 kD, 11.1 kD, 11.6 kD, 13.8 kl), 14.0 kD are dependent of the tyrosine-phosphorylation of CagA while the changes of 4 proteins expression at m/z of 4.71 kD, 4.73 kD, 6.5 kD and 6.7 kD are independent of the tyrosine-phosphorylation of CagA.3. Expression differences of 3 proteins at m/z of 4.2 kD, 8.2 kD and 9.1 kD are caused by activation of Erk/MAPK signaling pathway.4. In all, our results provide new targets for further understanding of the biological function and pathogenesis of CagA. |
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