Dataset Information

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

ABSTRACT: GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.

INSTRUMENT(S): ZenoTOF 7600

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ryan A. Flynn Benjamin A. Garcia

PROVIDER: MSV000093213 | MassIVE | Tue Oct 31 20:32:00 GMT 2023

REPOSITORIES: MassIVE

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Publications

The modified RNA base acp<sup>3</sup>U is an attachment site for N-glycans in glycoRNA.

Xie Yixuan Y Chai Peiyuan P Till Nicholas A NA Hemberger Helena H Lebedenko Charlotta G CG Porat Jennifer J Watkins Christopher P CP Caldwell Reese M RM George Benson M BM Perr Jonathan J Bertozzi Carolyn R CR Garcia Benjamin A BA Flynn Ryan A RA

Cell 20240821 19

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), ...[more]

PMID: 39173631

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Project description:Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat of cells called the glycocalyx. The aberrant changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA) has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate the application, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed GlycanDIA can distinguish glycan composition and isomers from N-glycans, O-glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N-glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N-glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation.

Project description:Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array we explored the interactions of glycans with C-type lectins, C-reactive protein and sera from T. suis infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties, but phosphorylcholine-modified mannose and N-acetylhexosaminesubstituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems, and also exhibit species-specific features distinguishing its glycome from those of other nematodes.Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array we explored the interactions of glycans with C-type lectins, C-reactive protein and sera from T. suis infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties, but phosphorylcholine-modified mannose and N-acetylhexosaminesubstituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems, and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

Dataset Information

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

Dataset's files

Publications

The modified RNA base acp<sup>3</sup>U is an attachment site for N-glycans in glycoRNA.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

Dataset's files

Publications

The modified RNA base acp&lt;sup&gt;3&lt;/sup&gt;U is an attachment site for N-glycans in glycoRNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

The modified RNA base acp<sup>3</sup>U is an attachment site for N-glycans in glycoRNA.