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Proceedings of the National Academy of... Oct 2023The family of GalNAc-Ts (GalNAcpolypeptide:N-Acetylgalactosaminyl transferases) catalyzes the first committed step in the synthesis of O-glycans, which is an abundant...
The family of GalNAc-Ts (GalNAcpolypeptide:N-Acetylgalactosaminyl transferases) catalyzes the first committed step in the synthesis of O-glycans, which is an abundant and biologically important protein modification. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence a broad array of biological functions. How site-specific O-glycans regulate biology is unclear. Compiling in vivo O-glycosites would be an invaluable step in determining the function of site-specific O-glycans. We integrated chemical and enzymatic conditions that cleave O-glycosites, a higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation mass spectrometry (MS) workflow and software to study nine mouse tissues and whole blood. We identified 2,154 O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and shared functions as classified by Gene Ontology terms. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We examined the Galnt2-null mouse model, which phenocopies congenital disorder of glycosylation involving GALNT2 and revealed a network of glycoproteins that lack GalNAc-T2-specific O-glycans. The known direct and indirect functions of these glycoproteins appear consistent with the complex metabolic phenotypes observed in the Galnt2-null animals. Through this study and interrogation of databases and the literature, we have compiled an atlas of experimentally identified mouse O-glycosites consisting of 2,925 O-glycosites from 758 glycoproteins.
Topics: Animals; Mice; Glycosylation; Glycoproteins; Proteome; Metabolic Diseases; Polysaccharides; Polypeptide N-acetylgalactosaminyltransferase
PubMed: 37862385
DOI: 10.1073/pnas.2303703120 -
Glycobiology Dec 2023N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (i) biosynthesis of a... (Review)
Review
N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (i) biosynthesis of a lipid-linked oligosaccharide (LLO), and (ii) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N-glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided an insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs.
Topics: Glycosylation; Hexosyltransferases; Lipopolysaccharides; Polysaccharides; Glycosyltransferases
PubMed: 37399117
DOI: 10.1093/glycob/cwad053 -
Analytical and Bioanalytical Chemistry Jul 2024Glycans participate in a vast number of recognition systems in diverse organisms in health and in disease. However, glycans cannot be sequenced because there is no... (Review)
Review
Glycans participate in a vast number of recognition systems in diverse organisms in health and in disease. However, glycans cannot be sequenced because there is no sequencer technology that can fully characterize them. There is no "template" for replicating glycans as there are for amino acids and nucleic acids. Instead, glycans are synthesized by a complicated orchestration of multitudes of glycosyltransferases and glycosidases. Thus glycans can vary greatly in structure, but they are not genetically reproducible and are usually isolated in minute amounts. To characterize (sequence) the glycome (defined as the glycans in a particular organism, tissue, cell, or protein), glycosylation pathway prediction using in silico methods based on glycogene expression data, and glycosylation simulations have been attempted. Since many of the mammalian glycogenes have been identified and cloned, it has become possible to predict the glycan biosynthesis pathway in these systems. By then incorporating systems biology and bioprocessing technologies to these pathway models, given the right enzymatic parameters including enzyme and substrate concentrations and kinetic reaction parameters, it is possible to predict the potentially synthesized glycans in the pathway. This review presents information on the data resources that are currently available to enable in silico simulations of glycosylation and related pathways. Then some of the software tools that have been developed in the past to simulate and analyze glycosylation pathways will be described, followed by a summary and vision for the future developments and research directions in this area.
Topics: Glycosylation; Computer Simulation; Polysaccharides; Animals; Humans; Software; Glycosyltransferases
PubMed: 38748247
DOI: 10.1007/s00216-024-05331-8 -
International Journal of Molecular... Oct 2023Apolipoprotein-CIII (apo-CIII) is involved in triglyceride-rich lipoprotein metabolism and linked to beta-cell damage, insulin resistance, and cardiovascular disease....
Apolipoprotein-CIII (apo-CIII) is involved in triglyceride-rich lipoprotein metabolism and linked to beta-cell damage, insulin resistance, and cardiovascular disease. Apo-CIII exists in four main proteoforms: non-glycosylated (apo-CIII), and glycosylated apo-CIII with zero, one, or two sialic acids (apo-CIII, apo-CIII and apo-CIII). Our objective is to determine how apo-CIII glycosylation affects lipid traits and type 2 diabetes prevalence, and to investigate the genetic basis of these relations with a genome-wide association study (GWAS) on apo-CIII glycosylation. We conducted GWAS on the four apo-CIII proteoforms in the DiaGene study in people with and without type 2 diabetes ( = 2318). We investigated the relations of the identified genetic loci and apo-CIII glycosylation with lipids and type 2 diabetes. The associations of the genetic variants with lipids were replicated in the Diabetes Care System ( = 5409). Rs4846913-A, in the -gene, was associated with decreased apo-CIII. This variant was associated with increased high-density lipoprotein cholesterol and decreased triglycerides, while high apo-CIII was associated with raised high-density lipoprotein-cholesterol and triglycerides. Rs67086575-G, located in the -gene, was associated with decreased apo-CIII and with hypertriglyceridemia. In line, apo-CIII was associated with low triglycerides. On a genome-wide scale, we confirmed that the -gene plays a major role i -glycosylation of apolipoprotein-CIII, with subsequent associations with lipid parameters. We newly identified the / region, in the literature previously associated with hypertriglyceridemia, as involved in apolipoprotein-CIII sialylation and hypertriglyceridemia. These results link genomics, glycosylation, and lipid metabolism, and represent a key step towards unravelling the importance of -glycosylation in health and disease.
Topics: Humans; Apolipoprotein C-III; Apolipoproteins C; Diabetes Mellitus, Type 2; Glycosylation; Genome-Wide Association Study; Triglycerides; Hypertriglyceridemia; Hyperlipidemias; Cholesterol, HDL; Receptors, Cytoplasmic and Nuclear; Vesicular Transport Proteins; Cytoskeletal Proteins; Adaptor Proteins, Signal Transducing
PubMed: 37834292
DOI: 10.3390/ijms241914844 -
Frontiers in Bioscience (Landmark... Nov 2023Glycosylation is one of the most common post-translational modifications of proteins across all kingdoms of life. Diverse monosaccharides and polysaccharides can be... (Review)
Review
Glycosylation is one of the most common post-translational modifications of proteins across all kingdoms of life. Diverse monosaccharides and polysaccharides can be attached to a range of amino acid residues generating N-glycosylation, O-glycosylation, C-glycosylation, S-glycosylation, as well as P-glycosylation. The functions of the eukaryotic glycosylation system during protein folding in the endoplasmic reticulum (ER) and Golgi are well-studied. Increasing evidence in the recent decade has demonstrated the presence of oligosaccharyltransferases (OSTs) in bacteria and archaea. In particular, the oligosaccharyltransferase (PglB) of and oligosaccharyltransferase (PglL) enzyme of are the most characterized OSTs that catalyze bacterial N-linked glycosylation and O-linked glycosylation, respectively. Glycoprotein administered as glycoconjugate vaccines have been shown to be effective prophylactic to protect against numerous pathogenic bacteria. The chemical synthesis of glycoproteins is complex and expensive, which limits its application to the development of glycoconjugate vaccines. However, studies have demonstrated that the biosynthesis of glycoproteins is realizable by transferring PglB, a plasmid encoding a substrate protein, or PglL, a plasmid encoding genes for glycan synthesis to This strategy can be applied to the development of glycoconjugate vaccines using engineered host . This review summarizes the structure and mechanism of action of the bacterial OSTs, PglB and PglL, and discusses their potential application to glycoconjugate vaccine design.
Topics: Escherichia coli; Bacterial Proteins; Glycoconjugates; Glycoproteins; Bacteria; Vaccines
PubMed: 38062836
DOI: 10.31083/j.fbl2811305 -
Biomolecules Jul 2023Rheumatoid arthritis (RA) Is a highly prevalent autoimmune disease that affects the joints but also various other organs. The disease is characterized by autoantibodies...
Rheumatoid arthritis (RA) Is a highly prevalent autoimmune disease that affects the joints but also various other organs. The disease is characterized by autoantibodies that are often already observed pre-disease. Since the 1980s, it has been known that antibody glycosylation is different in RA as compared to control individuals. While the literature on glycosylation changes in RA is dominated by reports on serum or plasma immunoglobulin G (IgG), our recent studies have indicated that the glycosylation changes observed for immunoglobulin A (IgA) and total serum -glycome (TSNG) may be similarly prominent, and useful in differentiating between the RA patients and controls, or as a proxy of the disease activity. In this study, we integrated and compared the RA glycosylation signatures of IgG, IgA and TSNG, all determined in the pregnancy-induced amelioration of rheumatoid arthritis (PARA) cohort. We assessed the association of the altered glycosylation patterns with the disease, autoantibody positivity and disease activity. Our analyses indicated a common, composite glycosylation signature of RA that was independent of the autoantibody status.
Topics: Female; Pregnancy; Humans; Glycosylation; Arthritis, Rheumatoid; Autoantibodies; Immunoglobulin G; Immunoglobulin A
PubMed: 37509142
DOI: 10.3390/biom13071106 -
Molecular Genetics and Metabolism Aug 2023PMM2-CDG is the most prevalent type of congenital disorders of glycosylation (CDG). It is caused by pathogenic variants in the gene encoding phosphomannomutase 2 (PMM2),...
PMM2-CDG is the most prevalent type of congenital disorders of glycosylation (CDG). It is caused by pathogenic variants in the gene encoding phosphomannomutase 2 (PMM2), which converts mannose-6-phosphate to mannose-1-phosphate and thus activates this saccharide for further glycosylation processes. Defective glycosylation can lead to an abnormal accumulation of unfolded proteins in endoplasmic reticulum (ER) and cause its stress. The ER is a key compartment for glycosylation, and its connection and communication with mitochondria has been described extensively in literature. Their crosstalk is important for cell proliferation, calcium homeostasis, apoptosis, mitochondrial fission regulation, bioenergetics, autophagy, lipid metabolism, inflammasome formation and unfolded protein response. Therefore, in the present study we posed a question, whether defective glycosylation leads to bioenergetic disruption. Our data reveal possible chronic stress in ER and activated unfolded protein response via PERK pathway in PMM2-CDG fibroblasts. Presumably, it leads to bioenergetic reorganization and increased assembly of respiratory chain complexes into supercomplexes together with suppressed glycolysis in PMM2-CDG patient cells. These changes cause alterations in Krebs cycle, which is tightly connected to electron transport system in mitochondria. In summary, we present data showing metabolic adaptation of cells to glycosylation defect caused by various pathogenic variants in PMM2.
Topics: Humans; Glycosylation; Congenital Disorders of Glycosylation; Phosphotransferases (Phosphomutases); Fibroblasts
PubMed: 37392701
DOI: 10.1016/j.ymgme.2023.107629 -
Analytical Chemistry Nov 2023Cell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific...
Cell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific information about glycoconjugate structures. Acquiring this information is essential for unraveling the functional role of glycans and for exploiting them as clinical targets. To specifically analyze the -glycoprotein forms expressed at the cell surface, we developed a C18 liquid chromatography (LC)-mass spectrometry (MS)-based glycoproteomics method in combination with highly specific cell surface protein labeling and enrichment using a biotin label. The surface-specificity of the method was validated by MS-based proteomics of subcellular component marker proteins. Using the human keratinocytes N/TERT-1 as a model system, we identified and quantified the glycosylation of hundreds of cell surface -glycosylation sites. This approach allowed us to study the glycoforms present at the functional relevant cell surface, omitting immaturely glycosylated proteins present in the secretory pathway. Interestingly, the different stages of -glycan processing at individual sites displayed at the cell surface were found to correlate with their accessibility for ER-residing processing enzymes, as investigated through molecular dynamics simulations. Using the new approach, we compared -glycosylation sites of proteins expressed on the cell surface to their counterparts in a total cell lysate, showing profound differences in glycosylation between the subcellular components and indicating the relevance of the method for future studies in understanding contextual glycan functions.
Topics: Humans; Glycosylation; Glycoproteins; Mass Spectrometry; Polysaccharides
PubMed: 37956981
DOI: 10.1021/acs.analchem.3c03626 -
JACS Au May 2024Most of the proteins in the circulation are -glycosylated, shaping together the total blood -glycome (TBNG). Glycosylation is known to affect protein function,... (Review)
Review
Most of the proteins in the circulation are -glycosylated, shaping together the total blood -glycome (TBNG). Glycosylation is known to affect protein function, stability, and clearance. The TBNG is influenced by genetic, environmental, and metabolic factors, in part epigenetically imprinted, and responds to a variety of bioactive signals including cytokines and hormones. Accordingly, physiological and pathological events are reflected in distinct TBNG signatures. Here, we assess the specificity of the emerging disease-associated TBNG signatures with respect to a number of key glycosylation motifs including antennarity, linkage-specific sialylation, fucosylation, as well as expression of complex, hybrid-type and oligomannosidic -glycans, and show perplexing complexity of the glycomic dimension of the studied diseases. Perspectives are given regarding the protein- and site-specific analysis of -glycosylation, and the dissection of underlying regulatory layers and functional roles of blood protein -glycosylation.
PubMed: 38818049
DOI: 10.1021/jacsau.4c00043 -
BMC Cancer Dec 2023Pancreatic cancer (PC) is a malignant tumor with extremely poor prognosis, exhibiting resistance to chemotherapy and immunotherapy. Nowadays, it is ranked as the third...
BACKGROUND
Pancreatic cancer (PC) is a malignant tumor with extremely poor prognosis, exhibiting resistance to chemotherapy and immunotherapy. Nowadays, it is ranked as the third leading cause of cancer-related mortality. Glycation is a common epigenetic modification that occurs during the tumor transformation. Many studies have demonstrated a strong correlation between glycation modification and tumor progression. However, the expression status of glycosylation-related genes (GRGs) in PC and their potential roles in PC microenvironment have not been extensively investigated.
METHOD
We systematically integrated RNA sequencing data and clinicopathological parameters of PC patients from TCGA and GTEx databases. A GRGs risk model based on glycosylation related genes was constructed and validated in 60 patients from Pancreatic biobank via RT-PCR. R packages were used to analyze the relationships between GRGs risk scores and overall survival (OS), tumor microenvironment, immune checkpoint, chemotherapy drug sensitivity and tumor mutational load in PC patients. Panoramic analysis was performed on PC tissues. The function of B3GNT8 in PC was detected via in vitro experiments.
RESULTS
In this study, we found close correlations between GRGs risk model and PC patients' overall survival and tumor microenvironment. Multifaceted predictions demonstrated the low-risk cohort exhibits superior OS compared to high-risk counterparts. Meanwhile, the low-risk group was characterized by high immune infiltration and may be more sensitive to immunotherapy or chemotherapy. Panoramic analysis was further confirmed a significant relationship between the GRGs risk score and both the distribution of PC tumor cells as well as CD8 + T cell infiltration. In addition, we also identified a unique glycosylation gene B3GNT8, which could suppress PC progression in vitro and in vivo.
CONCLUSION
We established a GRGs risk model, which could predict prognosis and immune infiltration in PC patients. This risk model may provide a new tool for PC precision treatment.
Topics: Humans; Prognosis; Glycosylation; Pancreatic Neoplasms; Pancreas; Immunotherapy; Tumor Microenvironment
PubMed: 38097951
DOI: 10.1186/s12885-023-11725-1