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Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and the Tn Antigen in Human Cancer Cells

ABSTRACT: Proteins modified with O-GlcNAc and O-GalNAc (the Tn antigen) play critically important roles in many physiological and pathological processes. Comprehensive and site-specific analysis of proteins modified with O-GlcNAc and the Tn antigen advances our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging to distinguish them because of the same chemical composition (identical molecular weights) and being localized on the same amino acid residues (S/T). Here, an effective method is developed through the integration of metabolic labeling, chemical and enzymatic reactions, and MS-based proteomics to simultaneously identify and distinguish glycoproteins modified with O-GlcNAc and the Tn antigen. In biologically duplicate experiments, 537 O-GlcNAcylated proteins and 103 glycoproteins modified with the Tn antigen were identified site-specifically in human cancer cells. Among O-GlcNAcylated proteins only in Jurkat cells, those involved in HTLV-1 infection were highly enriched, indicating that O-GlcNAcylation may play a role in regulating cellular response to the retrovirus. Glycoproteins in the spliceosome and with the nuclear localization signal (NLS) are also overrepresented. However, the O-GlcNAcylated proteins identified in all three cell lines were highly enriched with transcription and RNA binding functions. For glycoproteins with the Tn antigen, while many of them in Jurkat cells were highly enriched in the extracellular exosome, those in MCF7 and K562 cells were extensively found in the ER. The current method enables us to simultaneously analyze and unambiguously distinguish glycoproteins with O-GlcNAc and the Tn antigen, and it can be extensively applied for glycoscience research.

INSTRUMENT(S): LTQ Orbitrap Elite

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

SUBMITTER: Ronghu Wu

PROVIDER: MSV000088088 | MassIVE |

REPOSITORIES: MassIVE

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Project description:The O-N-acetyl-β-D-glucosaminylation, termed O-GlcNAcylation, is an atypical glycosylation corresponding to the transfer of a unique saccharide, the N-acetyl-β-D-glucosamine, on the hydroxyl group of serine and threonine amino acids of nuclear, cytosolic and mitochondrial proteins. Because of its dynamism, its reversibility and the swiftness of GlcNAc moieties transfer, O-GlcNAcylation is akin to phosphorylation and it is able to respond rapidly to both extracellular and intracellular demands. This post-translational modification is highly represented on intracellular proteins (in particular cytosolic, nuclear and mitochondrial proteins), and is involved in almost if not all cellular processes. O-GlcNAcylation is nowadays clearly associated with the etiology of several acquired diseases, in particular diabetes, neuro-degenerative disorders, cardiovascular diseases or cancer. The O-GlcNAcylation rapidly emerged as a major cellular mechanism which compete with phosphorylation in terms of modified proteins and the importance in cellular physiology. The O-GlcNAcylated sites could also correspond to phosphorylated ones; many proteins are modified by both O-GlcNAc and phosphates groups, and these two post-translational modifications could compete to the same or at neighboring sites. However, despite the undeniable role of O-GlcNAcylation in the modulation of almost all cellular processes, its precise function remains still misunderstood, mainly because of the unknown position of O-GlcNAcylation on specific domains of proteins. Thus, the precise localization of O-GlcNAcylated sites remains an indispensable prerequisite for the fine understanding of its biological function. However, mapping the O-GlcNAcylated sites remains laborious, but challenging, because of (i) the low stoichiometry of O-GlcNAcylation; (ii) the ion suppression of the modified peptide by the unmodified peptide present in larger excess; and (iii) the labile β bound between serine or threonine and the O-GlcNAc moiety which is broken during the CID (Collision-Induced Dissociation) fragmentation process, leading to loss of site information. To respond to this biological demand and to overcome these difficulties, enrichment of O-GlcNAc modified proteins and the use of other fragmentation processes like ECD (Electron Capture Dissociation), ETD (Electron Transfer Dissociation), or HCD (High-energy Collisional Dissociation), able to limit the O-GlcNAc loss during the fragmentation, are crucial. Our strategy was an alternative, specific, efficient and selective purification of O-GlcNAc bearing proteins from skeletal muscle cells by the use of the Click chemistry methodology. To extensively map O-GlcNAc sites on proteins, we proposed herein an intensive fractionation of the muscle cell proteome according to solubility, hydrophobicity and isoelectric point of proteins. The method of Click chemistry was achieved on each enriched-fraction containing proteins of interest.

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Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and the Tn Antigen in Human Cancer Cells

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