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Tailor-Made Detection of Individual Phosphorylated and Non-Phosphorylated EPIYA-Motifs of Helicobacter pylori Oncoprotein CagA

Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstraße 5, D-91058 Erlangen, Germany
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Cancers 2019, 11(8), 1163; https://doi.org/10.3390/cancers11081163
Received: 25 May 2019 / Revised: 25 July 2019 / Accepted: 8 August 2019 / Published: 13 August 2019
(This article belongs to the Special Issue Helicobacter pylori Associated Cancer)
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Abstract

The gastric pathogen and carcinogen Helicobacter pylori (H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.
Keywords: CagA; cagPAI; dotblot; EPIYA motifs; Helicobacter pylori; signaling; type IV secretion; Abl and Src tyrosine kinases CagA; cagPAI; dotblot; EPIYA motifs; Helicobacter pylori; signaling; type IV secretion; Abl and Src tyrosine kinases
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Pachathundikandi, S.K.; Gutiérrez-Escobar, A.J.; Tegtmeyer, N. Tailor-Made Detection of Individual Phosphorylated and Non-Phosphorylated EPIYA-Motifs of Helicobacter pylori Oncoprotein CagA. Cancers 2019, 11, 1163.

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