Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

<jats:title>Abstract</jats:title><jats:p>The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, <jats:italic>Helicobacter pylori</jats:italic> (<jats:italic>H. pylori</jats:italic>) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that <jats:italic>H. pylori</jats:italic> 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 <jats:italic>H. pylori.</jats:italic> The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during <jats:italic>H. pylori</jats:italic> infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in <jats:italic>in vitro</jats:italic> cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in <jats:italic>in vitro</jats:italic> reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.</jats:p>