Journal
CHEMICAL SCIENCE
Volume 7, Issue 12, Pages 6995-7001Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c6sc02889f
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Funding
- NSFC [21402235]
- National Major Project of China [2012ZX09J1210801, 2012ZX09502-001-005]
- Deutsche Forschungsgemeinschaft [Exc114/2, JU270/17-1, GRK2062]
- European Molecular Biology Laboratory (EMBL)
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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 a anomer of rhamnose is attached to Arg32 of EF-P, demonstrating that the corresponding glycosyltransferase EarP inverts the sugar of its cognate substrate dTDP-beta-L-rhamnose. Based on this finding we describe the synthesis of an alpha-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-Arg(Rha) 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.
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