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.

Establishment of a gastric cancer subline with high metastatic potential using a novel microfluidic system

“…Among them, E-cadherin and Smad3 were further detected in protein expression levels by Western blotting. E-cadherin is a crucial cell adhesion molecule and is considered to be a decisive inhibitor for tumor metastasis122829. Smad3 is a key molecule of the TGF-β/Smad signaling pathway.…”

Bacterial serine protease HtrA as a promising new target for antimicrobial therapy?

“…We have recently identified the cleavage sites of H. pylori HtrA in E-cadherin. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern as preferentially cleaved by HtrA [28]. The results of our studies also suggest that the presence of calcium ions blocks HtrA-mediated cleavage by interfering with the accessibility of calcium-binding regions between the individual EC domains harboring the HtrA cleavage sites [29].…”

“…The mechanism by which C. jejuni opens tight junctions is yet unknown. For both pathogens, an HtrA-mediated transmigration process was observed [20, 21, 28], enabling bacterial contact with basolaterally expressed receptors, such as α5β1 integrins or fibronectin [53, 54], but also allowing the bacteria to directly interact with cells of the immune system. It is currently being investigated whether C. jejuni prefers the transcellular migration or paracellular route, or whether this pathogen combines two pathways to overcome the epithelial barrier [48].…”

Pathogenesis of Helicobacter pylori infection

“…The serine protease HtrA has been recently introduced as a novel secreted virulence factor, cleaving the tumor suppressor and adherens junction protein E‐cadherin. A novel study identified three signature motifs in E‐cadherin containing the [VITA]‐[VITA]‐x‐x‐D‐[DN] consensus sequence, which was preferentially cleaved by HtrA . However, research progress is mainly hampered by the lack of available htrA mutants.…”