title: Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA.
abstract: The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.
authors.affiliation: Cancer Cluster Salzburg, Department of Molecular Biology, University of Salzburg, Austria.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Perna
authors.affiliation: Philochem AG, Otelfingen, Switzerland.
authors.forename: Tim
authors.initials: T
authors.lastname: Fugmann
authors.affiliation: Division of Microbiology, University of Erlangen-Nuremberg, Germany.
authors.forename: Manja
authors.initials: M
authors.lastname: Böhm
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
authors.lastname: Jan Hiss
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
authors.forename: Sarah
authors.initials: S
authors.lastname: Haller
authors.affiliation: Cancer Cluster Salzburg, Department of Molecular Biology, University of Salzburg, Austria.
authors.forename: Camilla
authors.initials: C
authors.lastname: Götz
authors.affiliation: Division of Microbiology, University of Erlangen-Nuremberg, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Tegtmeyer
authors.affiliation: Cancer Cluster Salzburg, Department of Molecular Biology, University of Salzburg, Austria.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Hoy
authors.affiliation: Institute of Pathology, University of Erlangen-Nuremberg, Germany.|Institute of Pathology, University of Bern, Bern, Switzerland.
authors.forename: Tilman T
authors.initials: TT
authors.lastname: Rau
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
authors.forename: Dario
authors.initials: D
authors.lastname: Neri
authors.affiliation: Division of Microbiology, University of Erlangen-Nuremberg, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Backert
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
authors.forename: Gisbert
authors.initials: G
authors.lastname: Schneider
authors.affiliation: Cancer Cluster Salzburg, Department of Molecular Biology, University of Salzburg, Austria.
authors.forename: Silja
authors.initials: S
authors.lastname: Wessler
mesh_terms: D020816:Amino Acid Motifs; D015703:Antigens, CD; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015820:Cadherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D015703:Antigens, CD; D001426:Bacterial Proteins; C513384:CDH1 protein, human; D015820:Cadherins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D057057:Serine Proteases