|Budget Amount *help
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,900,000 (Direct Cost: ¥2,900,000)
Helicobacter pylori CagA is injected into the host cell and tyrosine-phosphorylated. We examined tyrosine-phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine-phosphorylation sites were determined using quadropolar time-of flight mass spectrometry analysis. Specific anti-pY CagA polyclonal and anti-CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Mass spectrometry identified five crucial tyrosine-phosphorylation sites of CagA at Tyr893. Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H.pylori (NCTC11637)-infected AGS cells. CagA protein also had an immunoreceptor tyrosine-based activation motif (ITAM)-like amino acid sequences in the 3' region of the cagA, EPIYATIx_<27>EIYATI, which closely resembled the ITAM. CagA proteins, (i) were localized to the 1% TritonX-100 resistant membrane fraction (lipid rafts), (ii) formed a cluster of phosphorylated CagA protein complexes, (iii) associated with tyrosine-phosphorrylated GIT1/Cat1 (G protein-coupled receptor kinase-interactor 1/ Cool-associated, tyrosine-phosphorylated 1), substrate molecules of receptor type protein-tyrosine phosphatase, (RPTPζ/β), which is the receptor of VacA and (iv) were involved in a delay and negative regulation of VacA-induced signal. Immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine-phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. These findings suggest an important role for CagA containing ITAM-like sequences in the pathogenesis of H.pylori-related disease.