Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first ArgRha specific antibody

Li, Xiang; Krafczyk, Ralph; Macošek, Jakub; Li, Yu-Lei; Zou, Yan; Simon, Bernd; Pan, Xing; Wu, Qiu-Ye; Yan, Fang; Li, Shan; Hennig, Janosch; Jung, Kirsten; Lassak, Jürgen und Hu, Hong-Gang (2016): Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first ArgRha specific antibody. In: Chemical Science, Bd. 7, Nr. 12: S. 6695-7001

Abstract

A previously discovered posttranslational modification strategy – arginine rhamnosylation – is essential for elongation factor P (EF-P) dependent rescue of polyproline stalled ribosomes in clinically relevant species such as Pseudomonas aeruginosa and Neisseria meningitidis. However, almost nothing is known about this new type of N-linked glycosylation. In the present study we used NMR spectroscopy to show for the first time that the α anomer of rhamnose is attached to Arg32 of EF-P, demonstrating that the corresponding glycosyltransferase EarP inverts the sugar of its cognate substrate dTDP-β-L-rhamnose. Based on this finding we describe the synthesis of an α-rhamnosylated arginine containing peptide antigen in order to raise the first anti-rhamnosyl arginine specific antibody (anti-ArgRha). Using ELISA and Western Blot analyses we demonstrated both its high affinity and specificity without any cross-reactivity to other N-glycosylated proteins. Having the anti-ArgRha at hand we were able to visualize endogenously produced rhamnosylated EF-P. Thus, we expect the antibody to be not only important to monitor EF-P rhamnosylation in diverse bacteria but also to identify further rhamnosyl arginine containing proteins. As EF-P rhamnosylation is essential for pathogenicity, our antibody might also be a powerful tool in drug discovery.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Biologie > Department Biologie I
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 2041-6539
• Allianz-/Nationallizenz: Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
• Sprache: Englisch