Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line

Nielsen, Mathias I.; Haan, Noortje de; Kightlinger, Weston; Ye, Zilu; Dabelsteen, Sally; Li, Minyan; Jewett, Michael C.; Bagdonaite, Ieva; Vakhrushev, Sergey Y.; Wandall, Hans H.

doi:10.1038/s41467-022-33806-8

Cited by 16 publications

(14 citation statements)

References 66 publications

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“…It is worth mentioning that synergetic glycoproteomic methodologies using ( O -glyco)proteases such as IMPa and StcE have successfully identified Tn-containing glycopeptides, providing information on glycosylation sites and occupancy. 45 , 46 However, detailed characterization of the O -glycan repertoire is not yet feasible when studying glycopeptides.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive O-Glycan Analysis by Porous Graphitized Carbon Nanoliquid Chromatography–Mass Spectrometry

Zhang,

Wang,

Wuhrer

et al. 2024

Anal. Chem.

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The diverse and unpredictable structures of O-GalNActype protein glycosylation present a challenge for its structural and functional characterization in a biological system. Porous graphitized carbon (PGC) liquid chromatography (LC) coupled to mass spectrometry (MS) has become one of the most powerful methods for the global analysis of glycans in complex biological samples, mainly due to the extensive chromatographic separation of (isomeric) glycan structures and the information delivered by collision induced fragmentation in negative mode MS for structural elucidation. However, current PGC-based methodologies fail to detect the smaller glycan species consisting of one or two monosaccharides, such as the Tn (single GalNAc) antigen, which is broadly implicated in cancer biology. This limitation is caused by the loss of small saccharides during sample preparation and LC. Here, we improved the conventional PGC nano-LC-MS/MS-based strategy for O-glycan analysis, enabling the detection of truncated O-glycan species and improving isomer separation. This was achieved by the implementation of 2.7 μm PGC particles in both the trap and analytical LC columns, which provided an enhanced binding capacity and isomer separation for Oglycans. Furthermore, a novel mixed-mode PGC-boronic acid-solid phase extraction during sample preparation was established to purify a broad range of glycans in an unbiased manner, including the previously missed mono-and disaccharides. Taken together, the optimized PGC nano-LC-MS/MS platform presents a powerful component of the toolbox for comprehensive O-glycan characterization.

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Section: Resultsmentioning

confidence: 99%

Comprehensive O-Glycan Analysis by Porous Graphitized Carbon Nanoliquid Chromatography–Mass Spectrometry

Zhang,

Wang,

Wuhrer

et al. 2024

Anal. Chem.

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“…The monosaccharides linked to Ser and Thr residues in humans are mainly GalNAc and GlcNAc. 1087 , 1088 , 1089 GalNAc‐type O‐glycosylation is present on extracellular and secreted glycoproteins, such as mucins, 1090 , 1091 and this type of O‐glycosylation is initiated in the Golgi apparatus and regulated by up to 20 GalNActransferases (GALNTs), of which 15 isozymes have been demonstrated to be active enzymes. 1091 , 1092 GALNTs exhibit some specificity, but there is no specific amino acid motif for recognition in substrate proteins.…”

Section: Glycosylationmentioning

confidence: 99%

“…GlcNAc-β-Asn OST complex (STT3A/ STT3B) N-X-T/S, X≠P N-G; N-X-C/V, X≠P None Protein stability, 1118 protein folding and quality control, 1119 self/nonself recognition, 1120 cell adhesion 1121 , immunotherapy, 1122 receptor activation and endocytosis, 1123 glycoediting and drug delivery. 1124 O-glycosylation GalNAc-α-Ser/Thr GALNT1-20 Weak isoform specific motifs 1093 None O-glycan shielding is essential for secretion of an active protein, 1125 participates in immunological recognition of the immune system, 1082 protects membrane proteins from ectodomain shedding, 1090 increases the half-life of peptide hormones in circulation, 1126 modulates the interaction between viral proteins and host surface receptors. 1127 O-glycosylated mucins expressed at mucosal surfaces such as the respiratory and GI tract can form an effective barrier against pathogens.…”

Section: N-glycosylationmentioning

confidence: 99%

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Zhong

Xiao

Xie

et al. 2023

MedComm

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Protein posttranslational modifications (PTMs) refer to the breaking or generation of covalent bonds on the backbones or amino acid side chains of proteins and expand the diversity of proteins, which provides the basis for the emergence of organismal complexity. To date, more than 650 types of protein modifications, such as the most well‐known phosphorylation, ubiquitination, glycosylation, methylation, SUMOylation, short‐chain and long‐chain acylation modifications, redox modifications, and irreversible modifications, have been described, and the inventory is still increasing. By changing the protein conformation, localization, activity, stability, charges, and interactions with other biomolecules, PTMs ultimately alter the phenotypes and biological processes of cells. The homeostasis of protein modifications is important to human health. Abnormal PTMs may cause changes in protein properties and loss of protein functions, which are closely related to the occurrence and development of various diseases. In this review, we systematically introduce the characteristics, regulatory mechanisms, and functions of various PTMs in health and diseases. In addition, the therapeutic prospects in various diseases by targeting PTMs and associated regulatory enzymes are also summarized. This work will deepen the understanding of protein modifications in health and diseases and promote the discovery of diagnostic and prognostic markers and drug targets for diseases.

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“…The structural variation of O-glycans is controlled by the tissue and cell-specific repertoires of the glycosyltransferases involved in the formation of a highly variable core structure of mucin-type O-glycans, of their extensions by more or less elongated polylactosamine-type chains, and of peripheral modifications by α-linked monosaccharides [8]. Up to eight variants of mucin-type core structures were reported so far, which represent di-to tetrasaccharides formed starting from core-GalNAcs by adding Gal(1-3) (core 1), Gal (1)(2)(3) and GlcNAc(1-6) (core 2), GlcNAc(1-3) (core 3), and GlcNAc(1-3) and GlcNAc(1-6) (core 4) as major structures (Figure 1). Minor core-types (core 5 to core 8) were only rarely reported in specific tissues or cells or could, in the case of core 6, represent degradation products.…”

Section: Introductionmentioning

confidence: 99%

Site-Specific O-glycosylation of SARS-CoV-2 Spike Protein and Its Impact on Immune and Autoimmune Responses

Hanisch

2024

Cells

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The world-wide COVID-19 pandemic has promoted a series of alternative vaccination strategies aiming to elicit neutralizing adaptive immunity in the human host. However, restricted efficacies of these vaccines targeting epitopes on the spike (S) protein that is involved in primary viral entry were observed and putatively assigned to viral glycosylation as an effective escape mechanism. Besides the well-recognized N-glycan shield covering SARS-CoV-2 spike (S) proteins, immunization strategies may be hampered by heavy O-glycosylation and variable O-glycosites fluctuating depending on the organ sites of primary infection and those involved in immunization. A further complication associated with viral glycosylation arises from the development of autoimmune antibodies to self-carbohydrates, including O-linked blood group antigens, as structural parts of viral proteins. This outline already emphasizes the importance of viral glycosylation in general and, in particular, highlights the impact of the site-specific O-glycosylation of virions, since this modification is independent of sequons and varies strongly in dependence on cell-specific repertoires of peptidyl-N-acetylgalactosaminyltransferases with their varying site preferences and of glycan core-specific glycosyltransferases. This review summarizes the current knowledge on the viral O-glycosylation of the SARS-CoV-2 spike protein and its impact on virulence and immune modulation in the host.

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Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line

Cited by 16 publications

References 66 publications

Comprehensive O-Glycan Analysis by Porous Graphitized Carbon Nanoliquid Chromatography–Mass Spectrometry

Comprehensive O-Glycan Analysis by Porous Graphitized Carbon Nanoliquid Chromatography–Mass Spectrometry

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Site-Specific O-glycosylation of SARS-CoV-2 Spike Protein and Its Impact on Immune and Autoimmune Responses

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