Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.
Bottom Line: In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence.We found that v225d CagA interacts with SHP2 but not PAR1b.Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA.
Affiliation: Division of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.Show MeSH
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Mentions: CagA-PAR1b interaction has been shown to strengthen CagA-SHP2 interaction.13,14 Because v225d CagA did not bind to PAR1b, it was suggested that v225d CagA does not interact with SHP2 or, if it does, the binding ability would be very weak. To test this idea, we investigated the SHP2-binding activity of v225d CagA. COS-7 cells were co-transfected with a HA-tagged CagA expression vector and a FLAG-tagged SHP2 expression vector. Total cell lysates prepared were immunoprecipitated with an anti-HA antibody followed by immunoblotting with an anti-FLAG antibody. The results indicate that v225d CagA undergoes tyrosine phosphorylation and binds to SHP2 in cells overexpressing both proteins (Fig.3a). Next we investigated whether v225d CagA binds to endogenous SHP2. AGS human gastric epithelial cells were transfected with a HA-tagged CagA expression vector and then total cell lysates prepared were immunoprecipitated with an anti-HA antibody. Consistently, endogenous SHP2 was also co-immunoprecipitated with v225d CagA. However, the amount of SHP2 interacting with v225d CagA was much smaller than that with ABCCC CagA (Fig.3b).
Affiliation: Division of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.