Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains

BMC Microbiology

Research Article

Heinrich Sticht¹  Judith Lind²  Steffen Backert²  Nicole Tegtmeyer²  Rebecca Hoffmann³  Jutta Eichler³  Yoshio Yamaoka⁴  Guillermo I. Perez-Perez⁵  Javier Torres⁶ 

[1] Bioinformatics, Institute for Biochemistry, Friedrich Alexander University Erlangen-Nuremberg, Fahrstrasse 17, D-91054, Erlangen, Germany;Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstr. 5, D-91058, Erlangen, Germany;Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nuremberg, Schuhstraße 19, D-91052, Erlangen, Germany;Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu, Japan;Department of Medicine and Microbiology, New York University, Langone Medical Centre, New York, USA;Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría del Instituto Mexicano del Seguro Social, Mexico City, México;

关键词: c-Abl;    c-Src;    CagA;    cag;    Dotblot;    EPIYA motifs;    Gastric cancer;    Helicobacter pylori;    Signaling;    Type IV secretion;    T4SS;    Tyrosine kinases;   

DOI  :  10.1186/s12866-016-0820-6

 received in 2015-06-24, accepted in 2016-05-19,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundHighly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming.ResultsThis study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9–11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells.ConclusionsThe results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

【 授权许可】

CC BY   
© The Author(s). 2016