1. Cytotoxic necrotizing factor type 2 produced by pathogenic Escherichia coli deamidates a gln residue in the conserved G-3 domain of the rho family and preferentially inhibits the GTPase activity of RhoA and rac1
- Author
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Motoyuki Sugai, Eric Oswald, Akira Mogami, Minako Masuda, Frédéric Herault, Yasuhiko Horiguchi, Sylvie Y. Pérès, Kiyotaka Hatazaki, Hitoshi Komatsuzawa, Yoko Ueno, Hidekazu Suginaka, Hiroyuki Ohta, Hiroshima University, Okayama University, Unité mixte de recherche de microbiologie moléculaire, Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Osaka University [Osaka], and Hérault, Frédéric
- Subjects
rac1 GTP-Binding Protein ,RHOA ,GTPase-activating protein ,GTP' ,[SDV]Life Sciences [q-bio] ,Immunology ,Bacterial Toxins ,Blotting, Western ,Molecular Sequence Data ,RAC1 ,GTPase ,medicine.disease_cause ,Microbiology ,GTP Phosphohydrolases ,Substrate Specificity ,03 medical and health sciences ,medicine ,Escherichia coli ,Animals ,Amino Acid Sequence ,Peptide sequence ,[SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Cytotoxins ,Escherichia coli Proteins ,GTPase-Activating Proteins ,Biological activity ,Molecular biology ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Recombinant Proteins ,[SDV] Life Sciences [q-bio] ,Infectious Diseases ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Biochemistry ,COS Cells ,biology.protein ,Molecular and Cellular Pathogenesis ,Parasitology ,Electrophoresis, Polyacrylamide Gel ,[SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,rhoA GTP-Binding Protein - Abstract
Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122 RhoGAP . Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S -transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.
- Published
- 1999