title: Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains.
abstract: Highly 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.
  
  
  
  This 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.
  
  
  
  The 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.
authors: Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstr. 5, D-91058, Erlangen, Germany.
authors: Judith
authors: J
authors: Lind
authors: Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstr. 5, D-91058, Erlangen, Germany.
authors: Steffen
authors: S
authors: Backert
authors: Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nuremberg, Schuhstraße 19, D-91052, Erlangen, Germany.
authors: Rebecca
authors: R
authors: Hoffmann
authors: Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nuremberg, Schuhstraße 19, D-91052, Erlangen, Germany.
authors: Jutta
authors: J
authors: Eichler
authors: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu, Japan.
authors: Yoshio
authors: Y
authors: Yamaoka
authors: Department of Medicine and Microbiology, New York University, Langone Medical Centre, New York, USA.
authors: Guillermo I
authors: GI
authors: Perez-Perez
authors: Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría del Instituto Mexicano del Seguro Social, Mexico City, México.
authors: Javier
authors: J
authors: Torres
authors: Bioinformatics, Institute for Biochemistry, Friedrich Alexander University Erlangen-Nuremberg, Fahrstrasse 17, D-91054, Erlangen, Germany.
authors: Heinrich
authors: H
authors: Sticht
authors: Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstr. 5, D-91058, Erlangen, Germany. Nicole.Tegtmeyer@fau.de.
authors: Nicole
authors: N
authors: Tegtmeyer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D005753:Gastric Mucosa / Q000378:metabolism; D016481:Helicobacter Infections / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D013270:Stomach / Q000382:microbiology / Q000473:pathology; D013274:Stomach Neoplasms / Q000382:microbiology; D000069319:Type IV Secretion Systems
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000069319:Type IV Secretion Systems; C080866:cagA protein, Helicobacter pylori; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases
keywords: CagA; Dotblot; EPIYA motifs; Gastric cancer; Helicobacter pylori; Signaling; T4SS; Type IV secretion; Tyrosine kinases; c-Abl; c-Src; cagPAI