Issue 12, 2016

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

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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
30 Jun 2016
Accepted
20 Jul 2016
First published
21 Jul 2016
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2016,7, 6995-7001

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

X. Li, R. Krafczyk, J. Macošek, Y. Li, Y. Zou, B. Simon, X. Pan, Q. Wu, F. Yan, S. Li, J. Hennig, K. Jung, J. Lassak and H. Hu, Chem. Sci., 2016, 7, 6995 DOI: 10.1039/C6SC02889F

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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