ABSTRACT
The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.
Subject(s)
Glycosphingolipids/metabolism , Golgi Apparatus/metabolism , Golgi Matrix Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Autoantigens/genetics , Autoantigens/metabolism , Brefeldin A/pharmacology , Ceramides/metabolism , Cholera Toxin/pharmacology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Gene Expression , Glycosylation/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/genetics , Golgi Matrix Proteins/genetics , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Shiga Toxin/pharmacologyABSTRACT
Zinc finger protein 471 (ZNF471) is a member of the Krüppel-related domain zinc finger protein family, and has recently attracted attention because of its anti-cancer effects. N-glycosylation regulates expression and functions of the protein. This study aimed to investigate the effects of ZNF471 N-glycosylation on the proliferation, invasion, and docetaxel sensitivity of tongue squamous cell carcinoma (TSCC). It analyzed the expression, function, and prognostic significance of ZNF471 in TSCC using bioinformatics techniques such as gene differential expression analysis, univariate Cox regression analysis, functional enrichment analysis, and gene set enrichment analysis. Using site-specific mutagenesis, this study generated three mutant sites for ZNF471 N-glycosylation to determine the effect of N-glycosylation on ZNF471 protein levels and function. Quantitative real-time PCR, Western blot analysis, and immunohistochemistry tests confirmed the down-regulation of ZNF471 expression in TSCC. Low expression of ZNF471 is associated with poor prognosis of patients with TSCC. Overexpression of ZNF471 in vitro retarded the proliferation of TSCC cells and suppressed cell invasion and migration ability. Asparagine 358 was identified as a N-glycosylation site of ZNF471. Suppressing N-glycosylation of ZNF471 enhanced the protein stability and promoted the translocation of protein to the cell nucleus. ZNF471 binding to c-Myc gene promoter suppressed oncogene c-Myc expression, thereby playing the anti-cancer effect and enhancing TSCC sensitivity to docetaxel. In all, N-glycosylation of ZNF471 affects the proliferation, invasion, and docetaxel sensitivity of TSCC via regulation of c-Myc.
Subject(s)
Cell Proliferation , Docetaxel , Neoplasm Invasiveness , Proto-Oncogene Proteins c-myc , Repressor Proteins , Tongue Neoplasms , Female , Humans , Male , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Docetaxel/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Glycosylation/drug effects , Prognosis , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Tongue Neoplasms/pathology , Tongue Neoplasms/metabolism , Tongue Neoplasms/drug therapy , Tongue Neoplasms/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.
Subject(s)
Congenital Disorders of Glycosylation , Fibroblasts , Liposomes , Mannosephosphates , Phosphotransferases (Phosphomutases) , Humans , Glycosylation/drug effects , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/drug therapy , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Fibroblasts/metabolism , Fibroblasts/drug effects , Mannosephosphates/metabolism , Phosphotransferases (Phosphomutases)/genetics , Phosphotransferases (Phosphomutases)/metabolism , Phosphotransferases (Phosphomutases)/deficiency , Proteomics , Mannose/metabolismABSTRACT
INTRODUCTION: Fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG) is a rare autosomal recessive inborn error of metabolism characterized by a decreased flux through the salvage pathway of GDP-fucose biosynthesis due to a block in the recycling of L-fucose that exits the lysosome. FCSK-CDG has been described in 5 individuals to date in the medical literature, with a phenotype comprising global developmental delays/intellectual disability, hypotonia, abnormal myelination, posterior ocular disease, growth and feeding failure, immune deficiency, and chronic diarrhea, without clear therapeutic recommendations. PATIENT AND METHODS: In a so far unreported FCSK-CDG patient, we studied proteomics and glycoproteomics in vitro in patient-derived fibroblasts and also performed in vivo glycomics, before and after treatment with either D-Mannose or L-Fucose. RESULTS: We observed a marked increase in fucosylation after D-mannose supplementation in fibroblasts compared to treatment with L-Fucose. The patient was then treated with D-mannose at 850 mg/kg/d, with resolution of the chronic diarrhea, resolution of oral aversion, improved weight gain, and observed developmental gains. Serum N-glycan profiles showed an improvement in the abundance of fucosylated glycans after treatment. No treatment-attributed adverse effects were observed. CONCLUSION: D-mannose is a promising new treatment for FCSK-CDG.
Subject(s)
Congenital Disorders of Glycosylation , Fibroblasts , Mannose , Humans , Congenital Disorders of Glycosylation/drug therapy , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/pathology , Congenital Disorders of Glycosylation/metabolism , Mannose/metabolism , Fibroblasts/metabolism , Fibroblasts/drug effects , Male , Fucose/metabolism , Glycosylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Female , ProteomicsABSTRACT
Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.
Subject(s)
Archaeal Proteins/metabolism , Glycopeptides/metabolism , Glycoproteins/metabolism , Haloferax volcanii/metabolism , Amino Acid Sequence , Archaeal Proteins/chemistry , Biological Assay , Cell Shape/drug effects , Databases, Protein , Glycopeptides/chemistry , Glycoproteins/chemistry , Glycosylation/drug effects , Haloferax volcanii/drug effects , Mutation/genetics , Phenotype , Phylogeny , Proteomics , Sodium Chloride/pharmacologyABSTRACT
Glucose, the predominant carbohydrate in the human body, initiates nonenzymatic reactions in hyperglycemia, potentially leading to adverse biochemical interactions. This study investigates the interaction between glucose and Bovine Serum Albumin (BSA), along with the protective effects of Spirulina platensis PCC 7345 aqueous extract. Phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) in the extract were quantified using spectrophotometry. The extract's anti-glycation potential was assessed by analyzing its effects on albumin glycation, fluorescent advanced glycation end products (AGEs), thiol group oxidation, and ß-amyloid structure generation. Additionally, its antidiabetic potential was evaluated by measuring α-amylase and α-glucosidase enzyme inhibition. Results indicate that the Spirulina extract significantly mitigated ketoamine levels, fluorescence, and protein-carbonyl production induced by glucose, demonstrating a 67.81 % suppression of AGE formation after 28â days. Moreover, it effectively inhibited amyloid formation in BSA cross-linkages. These findings suggest the potential of S. platensis as an anti-glycation and antidiabetic agent, supporting its consideration for dietary inclusion to manage diabetes and associated complications.
Subject(s)
Glucose , Glycation End Products, Advanced , Serum Albumin, Bovine , Spirulina , Spirulina/chemistry , Serum Albumin, Bovine/metabolism , Serum Albumin, Bovine/chemistry , Glycosylation/drug effects , Animals , Cattle , Glucose/metabolism , Glycation End Products, Advanced/metabolism , Glycation End Products, Advanced/antagonists & inhibitors , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/isolation & purification , Water/chemistry , alpha-Amylases/antagonists & inhibitors , alpha-Amylases/metabolism , alpha-Glucosidases/metabolismABSTRACT
Osteoarthritis is more prevalent than any other form of arthritis and is characterized by the progressive mechanical deterioration of joints. Glucosamine, an amino monosaccharide, has been used for over fifty years as a dietary supplement to alleviate osteoarthritis-related discomfort. Silibinin, extracted from milk thistle, modifies the degree of glycosylation of target proteins, making it an essential component in the treatment of various diseases. In this study, we aimed to investigate the functional roles of glucosamine and silibinin in cartilage homeostasis using the TC28a2 cell line. Western blots showed that glucosamine suppressed the N-glycosylation of the gp130, EGFR, and N-cadherin proteins. Furthermore, both glucosamine and silibinin differentially decreased and increased target proteins such as gp130, Snail, and KLF4 in TC28a2 cells. We observed that both compounds dose-dependently induced the proliferation of TC28a2 cells. Our MitoSOX and DCFH-DA dye data showed that 1 µM glucosamine suppressed mitochondrial reactive oxygen species (ROS) generation and induced cytosol ROS generation, whereas silibinin induced both mitochondrial and cytosol ROS generation in TC28a2 cells. Our JC-1 data showed that glucosamine increased red aggregates, resulting in an increase in the red/green fluorescence intensity ratio, while all the tested silibinin concentrations increased the green monomers, resulting in decreases in the red/green ratio. We observed increasing subG1 and S populations and decreasing G1 and G2/M populations with increasing amounts of glucosamine, while increasing amounts of silibinin led to increases in subG1, S, and G2/M populations and decreases in G1 populations in TC28a2 cells. MTT data showed that both glucosamine and silibinin induced cytotoxicity in TC28a2 cells in a dose-dependent manner. Regarding endoplasmic reticulum stress, both compounds induced the expression of CHOP and increased the level of p-eIF2α/eIF2α. With respect to O-GlcNAcylation status, glucosamine and silibinin both reduced the levels of O-GlcNAc transferase and hypoxia-inducible factor 1 alpha. Furthermore, we examined proteins and mRNAs related to these processes. In summary, our findings demonstrated that these compounds differentially modulated cellular proliferation, mitochondrial and cytosol ROS generation, the mitochondrial membrane potential, the cell cycle profile, and autophagy. Therefore, we conclude that glucosamine and silibinin not only mediate glycosylation modifications but also regulate cellular processes in human chondrocytes.
Subject(s)
Chondrocytes , Glucosamine , Homeostasis , Kruppel-Like Factor 4 , Reactive Oxygen Species , Silybin , Glucosamine/pharmacology , Glucosamine/metabolism , Humans , Silybin/pharmacology , Glycosylation/drug effects , Chondrocytes/metabolism , Chondrocytes/drug effects , Homeostasis/drug effects , Reactive Oxygen Species/metabolism , Kruppel-Like Factor 4/metabolism , Cell Line , Cell Proliferation/drug effects , Mitochondria/metabolism , Mitochondria/drug effects , Cartilage/metabolism , Cartilage/drug effects , Oxidative Stress/drug effects , Osteoarthritis/metabolism , Osteoarthritis/drug therapyABSTRACT
Diabetes mellitus, characterized by dysregulated glucose metabolism, oxidative stress, and the formation of advanced glycation end products, poses a significant global health burden. In this study, we explored the potential of sorghum (Sorghum bicolor) seeds, known for their abundant phytochemical composition, as a natural remedy for diabetes and its associated damage. High-performance liquid chromatography/high-resolution mass spectrometry analysis revealed a remarkable phenolic richness in sorghum grains, including gallic acid, quercetin, and the predominant procyanidin B-1, with ecotype-specific variations in flavonoid distribution. Elemental analysis by ICP showed an abundance of macro-elements (Ca, K, Mg), trace elements (Fe, Mn, Si, Zn), and ultra-trace elements (B, Co, Cr, Cu, Mo, Se, V) essential for human health, supporting its therapeutic and nutritional potential. Additionally, the results demonstrated variable total phenolic contents (188-297 mg GAE/g dE) and total flavonoid contents (66-78 mg QE/g dE), with corresponding differences in antioxidant activities across the five ecotypes. Treatment with sorghum seed extract (SE1) significantly reduced oxidative stress markers, such as malondialdehyde (MDA)by 40% and hydrogen peroxide (H2O2) by 63%, in diabetic mice, compared to untreated diabetic controls. Moreover, sorghum extracts exhibited a remarkable increase in antioxidant enzyme activities, including a 50% increase in superoxide dismutase (SOD) activity and a 60% increase in glutathione peroxidase (GPx) activity, indicating their potential to bolster antioxidant defenses against diabetes-induced oxidative stress. These findings underscore the therapeutic potential of sorghum seeds in diabetes management and prevention, paving the way for the development of functional foods with enhanced health benefits.
Subject(s)
Antioxidants , Oxidative Stress , Plant Extracts , Seeds , Sorghum , Sorghum/chemistry , Oxidative Stress/drug effects , Seeds/chemistry , Animals , Mice , Plant Extracts/pharmacology , Plant Extracts/chemistry , Antioxidants/pharmacology , Hyperglycemia/drug therapy , Hyperglycemia/metabolism , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Glycation End Products, Advanced/metabolism , Flavonoids/pharmacology , Male , Glycosylation/drug effectsABSTRACT
The antiglycation mechanisms of three structurally different salvianolic acids (Sals) including salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and salvianolic acid C (Sal-C) were investigated using the bovine serum albumin (BSA)-fructose model. The results showed that the three compounds could inhibit the formation of glycation products, maintain protein structural stability, mitigate the development of amyloid fibrils and scavenge radicals. Notably, Sal-A possessed the highest anti-glycated activity compared with Sal-B and Sal-C. This may be related to the fact that Sal-A contained the most molecules of caffeic acid (Sal-A, Sal-B, and Sal-C possessing two, one, and zero caffeic acid units, respectively), and caffeic acid played a leading role in the antiglycation properties relative to Danshensu. Moreover, these compounds quenched the intrinsic fluorescence intensity of BSA in a static mode, with the binding constants in the order of Sal-A > Sal-B > Sal-C. Obviously, Sal-A possessed the strongest binding affinity among these compounds, which may be one of the reasons why it exhibited the optimal antiglycation capability. Furthermore, molecular docking demonstrated that the three Sals exerted protective effects on BSA by preventing glycation modification of lysine and arginine residues. These findings would provide valuable insights into the potential application of Sals for alleviating non-enzymatic glycation of protein.
Subject(s)
Benzofurans , Caffeic Acids , Lactates , Polyphenols , Serum Albumin, Bovine , Serum Albumin, Bovine/chemistry , Caffeic Acids/pharmacology , Caffeic Acids/chemistry , Glycosylation/drug effects , Polyphenols/pharmacology , Polyphenols/chemistry , Benzofurans/pharmacology , Benzofurans/chemistry , Lactates/pharmacology , Lactates/chemistry , Alkenes/pharmacology , Alkenes/chemistry , Animals , Glycation End Products, Advanced/chemistry , Glycation End Products, Advanced/metabolism , Cattle , Molecular Docking Simulation , DepsidesABSTRACT
BACKGROUND AND AIMS: HBV infection has been reported to trigger endoplasmic reticulum (ER) stress and initiate autophagy. However, how ER stress and autophagy influence HBV production remains elusive. Here, we studied the effect of tunicamycin (TM), an N-glycosylation inhibitor and ER stress inducer, on HBV replication and secretion and examined the underlying mechanisms. APPROACH AND RESULTS: Protein disulfide isomerase (an ER marker), microtubule-associated protein 1 light chain 3 beta (an autophagosome [AP] marker), and sequestosome-1 (a typical cargo for autophagic degradation) expression were tested in liver tissues of patients with chronic HBV infection and hepatoma cell lines. The role of TM treatment in HBV production and trafficking was examined in hepatoma cell lines. TM treatment that mimics HBV infection triggered ER stress and increased AP formation, resulting in enhanced HBV replication and secretion of subviral particles (SVPs) and naked capsids. Additionally, TM reduced the number of early endosomes and HBsAg localization in this compartment, causing HBsAg/SVPs to accumulate in the ER. Thus, TM-induced AP formation serves as an alternative pathway for HBsAg/SVP trafficking. Importantly, TM inhibited AP-lysosome fusion, accompanied by enhanced AP/late endosome (LE)/multivesicular body fusion, to release HBsAg/SVPs through, or along with, exosome release. Notably, TM treatment inhibited HBsAg glycosylation, resulting in impairment of HBV virions' envelopment and secretion, but it was not critical for HBsAg/SVP trafficking in our cell systems. CONCLUSIONS: TM-induced ER stress and autophagic flux promoted HBV replication and the release of SVPs and naked capsids through the AP-LE/MVB axis.
Subject(s)
Antiviral Agents/pharmacology , Carcinoma, Hepatocellular/metabolism , Endoplasmic Reticulum Stress , Hepatitis B virus/physiology , Hepatitis B, Chronic/physiopathology , Liver Neoplasms/metabolism , Tunicamycin/pharmacology , Virus Replication , Autophagosomes/drug effects , Autophagy/drug effects , Capsid , Cell Line, Tumor , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Endosomes/drug effects , Glycosylation/drug effects , Hepatitis B Surface Antigens/metabolism , Hepatitis B, Chronic/metabolism , Humans , Lysosomes/drug effects , Microtubule-Associated Proteins/metabolism , Multivesicular Bodies , Protein Disulfide-Isomerases/metabolism , Sequestosome-1 Protein/metabolism , VirionABSTRACT
Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Glycosylation/drug effects , Mannosidases/chemistry , Mannosidases/pharmacology , Animals , Bovine Virus Diarrhea-Mucosal Disease/drug therapy , Cattle , Cell Line , Dengue Virus/drug effects , Dogs , Glucosidases/metabolism , Humans , Madin Darby Canine Kidney Cells , Polysaccharides/metabolism , Secretory Pathway/drug effectsABSTRACT
The cleavage of the insulin receptor by ß-secretase 1 (BACE1) in the liver increases during diabetes, which contributes to reduce insulin receptor levels and impair insulin signaling. However, the precise signaling events that lead to this increased cleavage are unclear. We showed that BACE1 cleaves the insulin receptor in the early secretory pathway. Indeed, coimmunoprecipitation experiments reveal the interaction of the proforms of the two proteins. Moreover, fragments of insulin receptor are detected in the early secretory pathway and a mutated form of BACE1 that retains its prodomain cleaves an early secretory pathway-resident form of the insulin receptor. We showed that BACE1 proform levels are regulated by proteasome and/or lysosome-dependent degradation systems whose efficiencies are dependent on the O-GlcNacylation process. Our results showed that enhanced O-GlcNacylation reduces the efficiency of intracellular protein degradation systems, leading to the accumulation of the proform of BACE1 in the early secretory pathway where it cleaves the precursor of the insulin receptor. All these dysregulations are found in the livers of diabetic mice. In addition, we performed a screen of molecules according to their ability to increase levels of the insulin receptor at the surface of BACE1-overexpressing cells. This approach identified the aminosterol Claramine, which accelerated intracellular trafficking of the proform of BACE1 and increased autophagy. Both of these effects likely contribute to the reduced amount of the proform of BACE1 in the early secretory pathway, thereby reducing insulin receptor cleavage. These newly described properties of Claramine are consistent with its insulin sensitizing effect.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Cholestanes/pharmacology , Receptor, Insulin/metabolism , Spermine/analogs & derivatives , Animals , Diabetes Mellitus, Experimental/pathology , Disease Models, Animal , Endoplasmic Reticulum Stress/drug effects , Glycosylation/drug effects , HEK293 Cells , Hep G2 Cells , Humans , Liver/pathology , Models, Biological , Protein Binding/drug effects , Proteolysis/drug effects , Proteostasis/drug effects , Secretory Pathway/drug effects , Spermine/pharmacology , Ubiquitin/metabolism , Ubiquitination/drug effectsABSTRACT
Adoptive cellular therapy and its derivative, chimeric AgR T cell therapy, have achieved significant progress against cancer. Major barriers persist, however, including insufficient induction of cytotoxic T cells and exhaustion of tumor-infiltrating lymphocytes. In this study, we discovered a new role for 2-deoxy-d-glucose (2DG) in enhancing the antitumor activity of human T cells against NKG2D ligand-expressing tumor cells. Human T cells treated with 2DG upregulated the NK-specific transcription factors TOX2 and EOMES, thereby acquiring NK cell properties, including high levels of perforin/granzyme and increased sensitivity to IL-2. Notably, rather than inhibiting glycolysis, 2DG modified N-glycosylation, which augmented antitumor activity and cell surface retention of IL-2R of T cells. Moreover, 2DG treatment prevented T cells from binding to galectin-3, a potent tumor Ag associated with T cell anergy. Our results, therefore, suggest that modifying N-glycosylation of T cells with 2DG could improve the efficacy of T cell-based immunotherapies against cancer.
Subject(s)
Deoxyglucose/pharmacology , Immunity, Cellular/drug effects , Neoplasms/immunology , T-Lymphocytes/immunology , Glycosylation/drug effects , HMGB Proteins/immunology , Humans , Immunotherapy , Interleukin-2/immunology , K562 Cells , NK Cell Lectin-Like Receptor Subfamily K/immunology , Neoplasm Proteins/immunology , Neoplasms/pathology , Neoplasms/therapy , T-Box Domain Proteins/immunology , T-Lymphocytes/pathologyABSTRACT
Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.
Subject(s)
Bone Marrow Transplantation/adverse effects , CD8-Positive T-Lymphocytes/metabolism , Graft vs Host Disease/immunology , N-Acetylglucosaminyltransferases/metabolism , Receptors, Notch/metabolism , Animals , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/immunology , Disease Models, Animal , Feasibility Studies , Female , Flow Cytometry/methods , Glycosylation/drug effects , Humans , Leukosialin/metabolism , Ligands , Lymphocyte Activation/drug effects , Male , Mice , Sensitivity and Specificity , Sialomucins/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Stromal Cells/immunology , Stromal Cells/metabolism , Transplantation, Homologous/adverse effects , Up-RegulationABSTRACT
Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.
Subject(s)
Acetylglucosamine/metabolism , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , N-Acetylglucosaminyltransferases/metabolism , Animals , Biosynthetic Pathways/drug effects , Cells, Cultured , Cytokines/metabolism , Gene Expression/drug effects , Glucose/metabolism , Glycosylation/drug effects , Humans , Macrophages/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/drug effects , Monocytes/metabolism , Protein Processing, Post-Translational/drug effects , RAW 264.7 CellsABSTRACT
Background: Chemoresistance usually occurs in ovarian cancer. We aimed to explore the mechanisms of chemoresistance. Methods: Western blotting assay was used to detect the expression of GALNT14. Further cell function experiments were performed to investigate the effect of GALNT14 in ovarian cancer. Results: GALNT14 is significantly upregulated in ovarian cancer. Downregulation of GALNT14 significantly inhibits both apoptosis and ferroptosis of ovarian cancer cells. A further mechanism assay illustrated that downregulation of GALNT14 suppresses the activity of the mTOR pathway through modifying O-glycosylation of EGFR. Finally, an additive effect promoting cell death occurs with a combination of an mTOR inhibitor and cisplatin. Conclusion: Our study might provide a promising method to overcome cisplatin resistance for patients with ovarian cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cisplatin/pharmacology , Drug Resistance, Neoplasm/genetics , N-Acetylgalactosaminyltransferases/metabolism , Ovarian Neoplasms/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cisplatin/therapeutic use , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/metabolism , Female , Ferroptosis/drug effects , Ferroptosis/genetics , Gene Expression Regulation, Neoplastic , Glycosylation/drug effects , Humans , Middle Aged , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovary/pathology , Signal Transduction/drug effects , Signal Transduction/genetics , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Up-RegulationABSTRACT
The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.
Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/pharmacology , Biological Assay , Lectins/pharmacology , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Asparagine/chemistry , Asparagine/metabolism , Binding Sites , COVID-19/diagnosis , COVID-19/immunology , COVID-19/virology , Genes, Reporter , Glycosylation/drug effects , HEK293 Cells , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/immunology , SARS-CoV-2/drug effects , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization/drug effects , COVID-19 Drug TreatmentABSTRACT
Glycosyl phosphates are shown to be activated to stereospecific nucleophilic substitution reactions by precisely tailored bis-thiourea catalysts. Enhanced reactivity and scope is observed with phosphate relative to chloride leaving groups. Stronger binding (Km) to the H-bond donor and enhanced reactivity of the complex (kcat) enables efficient catalysis with broad functional group compatibility under mild, neutral conditions.
Subject(s)
Catalysis , Glycosylation , Phosphates/metabolism , Glycosylation/drug effects , Hydrogen Bonding , Lewis Acids/metabolism , Stereoisomerism , Thiourea/metabolismABSTRACT
α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation.
Subject(s)
Cell Proliferation/drug effects , Dystroglycans/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , Protective Agents/pharmacology , Animals , Basement Membrane/drug effects , Basement Membrane/virology , Body Fluids/drug effects , Body Fluids/virology , Cell Line , Glycosylation/drug effects , HEK293 Cells , Humans , Inflammation/drug therapy , Inflammation/virology , Influenza, Human/drug therapy , Influenza, Human/virology , Lung/drug effects , Lung/virology , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Viral Load/methodsABSTRACT
The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.