Essential Domain of Receptor Tyrosine Phosphatase β (RPTPβ) for Interaction with Helicobacter pylori Vacuolating Cytotoxin

Clustering of Helicobacter pylori VacA in Lipid Rafts, Mediated by Its Receptor, Receptor-Like Protein Tyrosine Phosphatase β, Is Required for Intoxication in AZ-521 Cells

“…Vacuolating activity was assessed using AZ-521 cells as previously described (44). Cells (1 ϫ 10 5 cells/well, 250 l) were grown in 48-well culture plates as monolayers for 24 h in a 5% CO 2 atmosphere at 37°C.…”

“…VacA was added, and cells were incubated at 37°C for the indicated times. To quantify vacuolating activity, the uptake of neutral red into vacuoles was determined as described previously (44).…”

“…We reported that two VacA proteins, termed m1VacA and m2VacA, which were defined by sequence differences in the middle of the molecules, interacted with target cells by binding to two types of receptor-like protein tyrosine phosphatases (RPTPs; i.e., RPTP␣ and RPTP␤), resulting in toxin internalization and vacuolation of the human gastric adenocarcinoma cell lines AZ-521 and G401 (12,30,43,44).…”

Direct binding of gangliosides to Helicobacter pylori vacuolating cytotoxin (VacA) neutralizes its toxin activity

“…VacA (s1m1) of H. pylori strain ATCC49503 was purified as previously described (Yahiro et al, 2004). The toxin was activated by incubating in HCl-PBS buffer, pH 2.0, at room temperature (RT) for 10 min before it was added to the cells.…”

“…Alexa Fluor dyes were purchased from Invitrogen (USA) and Alexa Fluor 488-cojugated antibody was purchased from -16 -Invitrogen (USA). Anti-VacA antibody was prepared as previously described (Yahiro et al, 2004). VacA was added to a final concentration of 240 nM and cells were incubated in EMEM containing 5 mM NH 4 Cl for an additional 3 h at 37C.…”