title: Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of Western-type Helicobacter pylori strains.
abstract: The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors--in particular various SH2-domain containing human proteins--thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9-11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA peptides, and that all of them recognized phosphotyrosines in mammalian proteins. These data are valuable in judicious application of commercial anti-phosphotyrosine antibodies and in characterization of CagA phosphorylation during infection and disease development.
authors.affiliation: Friedrich Alexander University Erlangen-Nuremberg, Department of Biology, Division of Microbiology, Erlangen, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Lind
authors.affiliation: Friedrich Alexander University Erlangen-Nuremberg, Department of Biology, Division of Microbiology, Erlangen, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Backert
authors.affiliation: Friedrich Alexander University Erlangen-Nuremberg, Department of Biology, Division of Microbiology, Erlangen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Pfleiderer
authors.affiliation: Division of Infectious Disease, Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Berg
authors.affiliation: Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamaoka
authors.affiliation: Friedrich Alexander University Erlangen-Nuremberg, Bioinformatics, Institute for Biochemistry, Erlangen, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sticht
authors.affiliation: Friedrich Alexander University Erlangen-Nuremberg, Department of Biology, Division of Microbiology, Erlangen, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Tegtmeyer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D005753:Gastric Mucosa / Q000378:metabolism; D016481:Helicobacter Infections / Q000378:metabolism*; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000502:physiology; D019000:Phosphotyrosine / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C080866:cagA protein, Helicobacter pylori; D019000:Phosphotyrosine