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Resolving the ?-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first Arg^Rha 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 ? anomer of rhamnose is attached to Arg32 of EF-P, demonstrating that the corresponding glycosyltransferase EarP inverts the sugar of its cognate substrate dTDP-?-l-rhamnose. Based on this finding we describe the synthesis of an ?-rhamnosylated arginine containing peptide antigen in order to raise the first anti-rhamnosyl arginine specific antibody (anti-Arg^Rha). 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.

SUBMITTER: Li X

PROVIDER: S-EPMC5363779 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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

Li Xiang X Krafczyk Ralph R Macošek Jakub J Li Yu-Lei YL Zou Yan Y Simon Bernd B Pan Xing X Wu Qiu-Ye QY Yan Fang F Li Shan S Hennig Janosch J Jung Kirsten K Lassak Jürgen J Hu Hong-Gang HG

Chemical science 20160721 12

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 ...[more]

PMID: 28451135

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Project description:Members of the genus Bifidobacterium are common inhabitants of the gastrointestinal tract of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their host. To extend our understanding of bifidobacterial carbohydrate utilisation, we investigated the molecular mechanisms by which various strains of Bifidobacterium breve metabolize four distinct α-glucose and/or α-galactose-containing oligosaccharides, namely raffinose, stachyose, melibiose and melezitose. Here we demonstrate that all B. breve strains examined possess the ability to utilise raffinose, stachyose and melibiose. However, the ability to metabolize melezitose was not ubiquitous for all tested B. breve strains. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified, yet being present only in those B. breve strains that were able to support growth on this carbohydrate. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

Dataset Information

Resolving the ?-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first Arg^Rha specific antibody.

Publications

Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first Arg<sup>Rha</sup> specific antibody.

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OmicsDI is part of the ELIXIR infrastructure

Dataset Information

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

Publications

Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first Arg<sup>Rha</sup> specific antibody.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Resolving the ?-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first Arg^Rha specific antibody.