TEO-GalNAc: Tag-free Chemoenzymatic Enrichment and Quantitative Mapping of Tn/T Antigen Sialylation Occupancy via Ketone-Oxime Chemistry
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Mingliang Ye
PROVIDER: PXD075239 | iProX | Wed Mar 04 00:00:00 GMT 2026
REPOSITORIES: iProX
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Project description:TEO-GalNAc is a chemoenzymatic strategy for tag-free enrichment and quantitative mapping of Tn/T O-GalNAc glycopeptides. Ketone-modified CMP-Sia is transferred by ST6GalNAc1 for selective tagging, followed by reversible oxime capture using hydroxylamine beads. Mild acid release enables tag-free recovery and quantitative assessment of sialylation occupancy on core GalNAc residues in complex samples.
2026-06-23 | PXD080093 |
Project description:Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser and Thr residues, is found on most solid tumors yet rarely detected in adult tissues: featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of the new vaccines, diagnostics, and therapeutics of cancers. Here, we report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn. This exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves N-termini of the Gal(13C6)-Tn-occupied Ser and Thr residues to yield site-containing glycopeptides. The enzymes C1GalT1 and OpeRATOR could be used concurrently in one-pot. The effectiveness of EXoO-Tn was evaluated by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, relative position in proteins, and mapped site-specific Tn-glycoproteome in different studies. Given the significance of Tn in cancers, EXoO-Tn is anticipated to have broad utilities in clinical study of cancers.
2022-02-15 | PXD014390 | Pride
Project description:Tumors are prone to immune escape by developing strategies to evade immunity. ST6GalNAc-I is an O-glycosyltransferase, which catalyzes the addition of sialic acid onto the first sugar GalNAc (Tn) and results in the formation of Neu5Acα2, 6GalNAc (STn) carbohydrate antigen. SialylTn plays an important role in tumor progression, angiogenesis, and metastasis. RNA sequence analysis was performed using genetically engineered LUAD mouse model tumors (KrasG12D/+; Trp53R172H/+; Ad-Cre (KPA) and KrasG12D/+; Ad-Cre (KA)). We have performed RNA sequence analysis using KPA tumors (N=3), KA tumors (N=3), and littermate control lung tissues (N=3). In comparison to normal lung tissues, several genes were significantly overexpressed in KPA and KA-derived LUAD tumors, including Meg3, Slc7a5, Cxcr1, Awat1, Kng2, Gjb3, Mfi2, and Dlk1. More specifically, St6galnac-I was significantly overexpressed in aggressive-type KPA tumor tissues compared to KA and normal lung tissues. Expression of ST6GalNAc-I is associated with tumor progression and angiogenesis by immunosuppression. Mechanistically, ST6GalNAc-I promotes tumor cell sialylation that impacts tumor angiogenesis by altering mucin MUC5AC. Similarly, ST6GalNAc-I regulates Nectin2 sialylation which leads to immunosuppression and tumor progression. Overall, our study defined that ST6GalNAc-I is a critical molecule for tumor cell sialylation for disease aggressiveness.
2025-04-15 | GSE290957 | GEO
Project description:Chronic inflammatory bowel disease (IBD) causes disability, suffering and risk of colon cancer in over 30 million people globally. Here, we investigate 3 kindreds of IBD with mutations in the N-acetylgalactosamide alpha-2,6-sialyltransferase 1/ST6GALNAC1 (ST6) glycosyltransferase gene that is uniquely expressed in the goblet cells (GCs). These mutations cause defective sialic acid (SA) conjugation, elimination of the onco-antigen S-Tn (Sialyl-Tn), and altered glycosylation of the MUC2 protein, a key component of intestinal mucus. Decreased MUC2 sialylation increases susceptibility to bacterial proteolytic degradation and compromises the gastrointestinal (GI) mucus barrier. Mice harboring the patient ST6 mutations recapitulate human colitis and reveal that defective sialylation causes dysbiosis, butyrate overproduction, and impaired GI stem cell proliferation during injury. Thus, this new genetic disease illustrates how sialylation controls host-microbe homeostasis and reveals several new potential approaches to IBD treatment.
2022-03-16 | GSE183821 | GEO
Project description:Chemoenzymatic labeling has greatly advanced O-GlcNAc proteomics, yet existing strategies using azide-modified donors (e.g., UDP-GalNAz) require multi-step click-based workflows that introduce substantial sample loss and limit sensitivity toward low-abundance targets. While recent one-step labeling with biotinylated UDP-GalNAc donors streamlines workflows, the irreversible avidin–biotin interaction prevents glycopeptide recovery and restricts site-specific analysis. To address these challenges, we developed a ketone-reversible O-GlcNAc enrichment (KRO-GlcNAc) strategy employing a ketone-functionalized UDP-GalNAc analog (UDP-GalNLev) for one-step enzymatic tagging. GalT1(Y289L) transfers the GalNLev moiety with >96% efficiency, eliminating intermediate reactions and minimizing handling-induced loss. Crucially, reversible oxime-based capture on hydroxylamine-functionalized beads followed by mild methoxyamine release not only overcomes the irreversible nature of reported ketone-hydroxylamine-biotin enrichment, but fundamentally address the broader limitations that originally necessitated the transition from ketone-based labeling to azide chemistry. Application of KRO-GlcNAc to HeLa cells identified 4,129 potential O-GlcNAcylation sites across 740 proteins, with ~30% being novel. Functional studies revealed that O-GlcNAcylation regulates nuclear-cytoplasmic shuttling of chromatin remodeler EP400, linking this modification to stress granule assembly. This platform thus provides a powerful and versatile tool for advancing O-GlcNAcylation research.
2026-01-06 | PXD072723 |
Project description:Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.
2025-11-06 | GSE282614 | GEO
Project description: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.
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Project description:Chronic inflammatory bowel disease (IBD) causes disability, suffering and risk of colon cancer in over 30 million people globally. Here, we investigate 3 kindreds of IBD with mutations in the N-acetylgalactosamide alpha-2,6-sialyltransferase 1/ST6GALNAC1 (ST6) glycosyltransferase gene that is uniquely expressed in the goblet cells (GCs). These mutations cause defective sialic acid (SA) conjugation, elimination of the onco-antigen S-Tn (Sialyl-Tn), and altered glycosylation of the MUC2 protein, a key component of intestinal mucus. Decreased MUC2 sialylation increases susceptibility to bacterial proteolytic degradation and compromises the gastrointestinal (GI) mucus barrier. Mice harboring the patient ST6 mutations recapitulate human colitis and reveal that defective sialylation causes dysbiosis, butyrate overproduction, and impaired GI stem cell proliferation during injury. Thus, this new genetic disease illustrates how sialylation controls host-microbe homeostasis and reveals several new potential approaches to IBD treatment.
2022-03-17 | PXD028239 | Pride
Project description:Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through the proteolytic cleavage of an exposed reactive centre loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localised Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterisation of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we firstly performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics (MD) simulations to study their impact on NE proteolysis. Importantly, we also identified novel O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 sites was experimentally observed and supported in silico by modelling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and -T3 abundantly expressed by liver and gallbladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG (rCBG) was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialo- (disialyl T) and asialo-glycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. MD substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on strategically-positioned and biologically-relevant CBG RCL glycans, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.
2023-12-05 | PXD046405 | Pride
Project description:GalNAc-transferase (GalNAc-T) isoforms modify distinct subsets of the O-glycoproteome and GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and T2) in liver cells, and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and T2. Differential transcriptomic analysis indicates that loss of function of a GalNAc-T induces specific transcriptional response. The non-redundant O-glycoproteome subsets for and the transcriptional responses for each isoform appeared to be related to different cellular processes, and for the GalNAc-T2 isoform supporting a role in lipid metabolism. The results demonstrate that GalNAc-Ts have non-redundant glycosylation functions, and that these may affect distinct cellular processes. The data provides a comprehensive resource for unique substrates for individual GalNAc-Ts. Our study provides a new view on the regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.
2015-09-30 | E-MTAB-3844 | biostudies-arrayexpress