RESUMO
Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9, simulating that found in a patient with CMS. As Gfpt1 exon 9 is exclusively expressed in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (1) UDP-HexNAc, CMP-NeuAc and protein O-GlcNAcylation were reduced in skeletal muscles; (2) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (3) markers of the unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of endoplasmic reticulum (ER) stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.
Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Músculo Esquelético , Dobramento de Proteína , Resposta a Proteínas não Dobradas , Animais , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Apoptose , Camundongos , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Especificidade de Órgãos , Proteína Forkhead Box O1/metabolismo , Técnicas de Introdução de Genes , Proteína 1 de Ligação a X-Box/metabolismo , Agregados Proteicos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
BACKGROUND: N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells. METHODS: Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity. RESULTS: Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases. CONCLUSIONS: The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity. GENERAL SIGNIFICANCE: Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.
Assuntos
Complexo de Golgi , N-Acetilglucosaminiltransferases , Humanos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Especificidade por Substrato , Complexo de Golgi/metabolismo , Complexo de Golgi/genética , Mutação , Polissacarídeos/metabolismo , Domínio Catalítico , GlicosilaçãoRESUMO
Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing and signaling mechanisms governing cancer cell stemness can provide insights into ameliorating cancer relapse and therapeutic resistance. Here, we provide convincing evidence that chronic metabolic stress triggered by hyaluronan production augments CSC-like traits and chemoresistance by partially impairing nucleotide sugar metabolism, dolichol lipid-linked oligosaccharide (LLO) biosynthesis and N-glycan assembly. Notably, preconditioning with either low-dose tunicamycin or 2-deoxy-D-glucose, which partially interferes with LLO biosynthesis, reproduced the promoting effects of hyaluronan production on CSCs. Multi-omics revealed characteristic changes in N-glycan profiles and Notch signaling activation in cancer cells exposed to mild glycometabolic stress. Restoration of N-glycan assembly with glucosamine and mannose supplementation and Notch signaling blockade attenuated CSC-like properties and further enhanced the therapeutic efficacy of cisplatin. Therefore, our findings uncover a novel mechanism by which tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.
Assuntos
Ácido Hialurônico , Resiliência Psicológica , Humanos , Glicosilação , Ácido Hialurônico/metabolismo , Recidiva Local de Neoplasia/metabolismo , Polissacarídeos/metabolismo , Suplementos Nutricionais , Células-Tronco Neoplásicas/metabolismoRESUMO
The dystrophin-glycoprotein complex connects the cytoskeleton with base membrane components such as laminin through unique O-glycans displayed on α-dystroglycan (α-DG). Genetic impairment of elongation of these glycans causes congenital muscular dystrophies. We previously identified that glycerol phosphate (GroP) can cap the core part of the α-DG O-glycans and terminate their further elongation. This study examined the possible roles of the GroP modification in cancer malignancy, focusing on colorectal cancer. We found that the GroP modification critically depends on PCYT2, which serves as cytidine 5'-diphosphate-glycerol (CDP-Gro) synthase. Furthermore, we identified a significant positive correlation between cancer progression and GroP modification, which also correlated positively with PCYT2 expression. Moreover, we demonstrate that GroP modification promotes the migration of cancer cells. Based on these findings, we propose that the GroP modification by PCYT2 disrupts the glycan-mediated cell adhesion to the extracellular matrix and thereby enhances cancer metastasis. Thus, the present study suggests the possibility of novel approaches for cancer treatment by targeting the PCYT2-mediated GroP modification.
Assuntos
Distroglicanas , Neoplasias , RNA Nucleotidiltransferases/metabolismo , Distroglicanas/genética , Distroglicanas/metabolismo , Glicerol/metabolismo , Glicerofosfatos , Humanos , Fosfatos/metabolismo , Polissacarídeos/metabolismo , Regulação para CimaRESUMO
AIMS: The number of cancer survivors with cardiovascular disease is increasing. However, the effect of cancer on body fluid regulation remains to be clarified. In this study, we evaluated body osmolyte and water imbalance in rats with hepatocellular carcinoma. MAIN METHODS: Wistar rats were administered diethylnitrosamine, a carcinogenic drug, to establish liver cancer. We analyzed tissue osmolyte and water content, and their associations with aldosterone secretion. KEY FINDINGS: Hepatocellular carcinoma rats had significantly reduced body mass and the amount of total body sodium, potassium, and water. However, these rats had significantly increased relative tissue sodium, potassium, and water content per tissue dry weight. Furthermore, these changes in sodium and water balance in hepatocellular carcinoma rats were significantly associated with increased 24-h urinary aldosterone excretion. Supplementation with 0.25% salt in drinking water improved body weight reduction associated with sodium and water retention in hepatocellular carcinoma rats, which was suppressed by treatment with spironolactone, a mineralocorticoid receptor antagonist. Additionally, the urea-driven water conservation system was activated in hepatocellular carcinoma rats. SIGNIFICANCE: These findings suggest that hepatocellular carcinoma induces body mass loss in parallel with activation of the water conservation system including aldosterone secretion and urea accumulation to retain osmolyte and water. The osmolyte and water retention at the tissue level may be a causative factor for ascites and edema formation in liver failure rats.
Assuntos
Aldosterona/urina , Carcinoma Hepatocelular/urina , Dietilnitrosamina/toxicidade , Neoplasias Hepáticas Experimentais/urina , Equilíbrio Hidroeletrolítico , Redução de Peso , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Ratos , Ratos Endogâmicos WKY , Receptores de Mineralocorticoides/metabolismo , Espironolactona/farmacologiaRESUMO
N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/química , Ensaios de Triagem em Larga Escala/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Polissacarídeos/química , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Corantes Fluorescentes , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.
Assuntos
Vesículas Extracelulares/metabolismo , Animais , Linhagem Celular Tumoral , Desoxiglucose/metabolismo , Dolicóis/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/ultraestrutura , Glicosilação , Lipídeos/química , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteínas de Membrana/metabolismo , Camundongos , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Infected or damaged tissues release multiple "alert" molecules such as alarmins and damage-associated molecular patterns (DAMPs) that are recognized by innate immune receptors, and induce tissue inflammation, regeneration, and repair. Recently, an extract from inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®, NTP) was found to induce infarct tolerance in mice receiving permanent ischemic attack to the middle cerebral artery. Likewise, we report herein that NTP prevented the neurite retraction in PC12 cells by nerve growth factor (NGF) deprivation. This effect was accompanied by interaction of Fyn with high-affinity NGF receptor TrkA. Sucrose density gradient subcellular fractionation of NTP-treated cells showed heretofore unidentified membrane fractions with a high-buoyant density containing Trk, B subunit of cholera toxin-bound ganglioside, flotillin-1 and Fyn. Additionally, these new membrane fractions also contained Toll-like receptor 4 (TLR4). Inhibition of TLR4 function by TAK-242 prevented the formation of these unidentified membrane fractions and suppressed neuroprotection by NTP. These observations indicate that NTP controls TrkA-mediated signaling through the formation of clusters of new membrane microdomains, thus providing a platform for crosstalk between neurotrophic and innate immune receptors. Neuroprotective mechanisms through the interaction with innate immune systems may provide novel mechanism for neuroprotection.
Assuntos
Imunidade Inata/efeitos dos fármacos , Polissacarídeos/metabolismo , Receptor Cross-Talk/efeitos dos fármacos , Animais , Gangliosídeos/metabolismo , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fator de Crescimento Neural/metabolismo , Neuritos/metabolismo , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Células PC12 , Fosforilação/efeitos dos fármacos , Polissacarídeos/imunologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Receptor Cross-Talk/imunologia , Receptor Cross-Talk/fisiologia , Receptor trkA/imunologia , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.
Assuntos
Fucose/análogos & derivados , Fucose/química , Fucosiltransferases/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Sítios de Ligação , Biotinilação , Química Click , Fucose/metabolismo , Fucosiltransferases/química , Fucosiltransferases/genética , Glicosilação , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMO
Fucosylation is a glycan modification critically involved in cancer and inflammation. Although potent fucosylation inhibitors are useful for basic and clinical research, only a few inhibitors have been developed. Here, we focus on a fucose analog with an alkyne group, 6-alkynyl-fucose (6-Alk-Fuc), which is used widely as a detection probe for fucosylated glycans, but is also suggested for use as a fucosylation inhibitor. Our glycan analysis using lectin and mass spectrometry demonstrated that 6-Alk-Fuc is a potent and general inhibitor of cellular fucosylation, with much higher potency than the existing inhibitor, 2-fluoro-fucose (2-F-Fuc). The action mechanism was shown to deplete cellular GDP-Fuc, and the direct target of 6-Alk-Fuc is FX (encoded by TSTA3), the bifunctional GDP-Fuc synthase. We also show that 6-Alk-Fuc halts hepatoma invasion. These results highlight the unappreciated role of 6-Alk-Fuc as a fucosylation inhibitor and its potential use for basic and clinical science.
Assuntos
Alcinos/farmacologia , Antineoplásicos/farmacologia , Carboidratos Epimerases/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Fucose/farmacologia , Guanosina Difosfato Fucose/biossíntese , Cetona Oxirredutases/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Alcinos/química , Antineoplásicos/química , Carboidratos Epimerases/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Fucose/química , Células HEK293 , Células HeLa , Humanos , Cetona Oxirredutases/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologiaRESUMO
Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.
Assuntos
Hexosaminas/biossíntese , Ácido Hialurônico/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , Animais , Feminino , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Hexosaminas/genética , Ácido Hialurônico/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Transferases de Grupos Nitrogenados/genética , Transferases de Grupos Nitrogenados/metabolismoRESUMO
Fucose, a terminal sugar in glycoconjugates, critically regulates various physiological and pathological phenomena, including cancer development and inflammation. However, there are currently no probes for efficient labeling and detection of this sugar. We chemically synthesized a novel series of alkynyl-fucose analogs as probe candidates and found that 7-alkynyl-fucose gave the highest labeling efficiency and low cytotoxicity. Among the fucose analogs, 7-alkynyl-fucose was the best substrate against all five fucosyltransferases examined. We confirmed its conversion to the corresponding guanosine diphosphate derivative in cells and found that cellular glycoproteins were labeled much more efficiently with 7-alkynyl-fucose than with an existing probe. 7-Alkynyl-fucose was detected in the N-glycan core by mass spectrometry, and 7-alkynyl-fucose-modified proteins mostly disappeared in core-fucose-deficient mouse embryonic fibroblasts, suggesting that this analog mainly labeled core fucose in these cells. These results indicate that 7-alkynyl-fucose is a highly sensitive and powerful tool for basic glycobiology research and clinical application for biomarker discovery.
Assuntos
Biomarcadores Tumorais/análise , Fucose/farmacologia , Sondas Moleculares/farmacologia , Polissacarídeos/análise , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fucose/análogos & derivados , Fucose/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Sondas Moleculares/síntese química , Sondas Moleculares/químicaRESUMO
O-GlcNAcylation is a reversible post-translational modification. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. More recent evidence indicates that regulation of O-GlcNAcylation is important for inflammatory diseases and tumorigenesis. In this study, we revealed that O-GlcNAcylation was increased in the colonic tissues of dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models. Moreover, the O-GlcNAcylation level was elevated in human CAC tissues compared with matched normal counterparts. To investigate the functional role of O-GlcNAcylation in colitis, we used OGA heterozygote mice, which have an increased level of O-GlcNAcylation. OGA(+/-) mice have higher susceptibility to DSS-induced colitis than OGA(+/+) mice. OGA(+/-) mice exhibited a higher incidence of colon tumors than OGA(+/+) mice. In molecular studies, elevated O-GlcNAc levels were shown to enhance the activation of NF-κB signaling through increasing the binding of RelA/p65 to its target promoters. We also found that Thr-322 and Thr352 in the p65-O-GlcNAcylation sites are critical for p65 promoter binding. These results suggest that the elevated O-GlcNAcylation level in colonic tissues contributes to the development of colitis and CAC by disrupting regulation of NF-κB-dependent transcriptional activity.
Assuntos
Colite/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica/genética , NF-kappa B/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Western Blotting , Transformação Celular Neoplásica/genética , Colite/metabolismo , Colite/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Processamento de Proteína Pós-TraducionalRESUMO
Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteínas de Homeodomínio/biossíntese , Esclerose Múltipla/genética , N-Acilesfingosina Galactosiltransferase/genética , Proteínas do Tecido Nervoso/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Galactosilceramidas/biossíntese , Galactosilceramidas/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Células HeLa , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Humanos , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , N-Acilesfingosina Galactosiltransferase/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/enzimologia , Oligodendroglia/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Proteínas de Peixe-ZebraAssuntos
Encéfalo/metabolismo , Metabolismo Energético , Glucose/metabolismo , Glicolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Colesterol/análogos & derivados , Colesterol/biossíntese , Colesterol/metabolismo , Glucosilceramidas/biossíntese , Glucosilceramidas/metabolismo , Glicerofosfolipídeos/metabolismo , Glicolipídeos/química , Glicosilação , Humanos , Microdomínios da Membrana/metabolismo , Camundongos , Ácidos Fosfatídicos/metabolismo , Esfingolipídeos/biossínteseRESUMO
The glycolipid Glc3Man9GlcNAc2-pyrophosphate-dolichol serves as the precursor for asparagine (N)-linked protein glycosylation in mammals. The biosynthesis of dolichol-linked oligosaccharides (DLOs) is arrested in low-glucose environments via unknown mechanisms, resulting in abnormal N-glycosylation. Here, we show that under glucose deprivation, DLOs are prematurely degraded during the early stages of DLO biosynthesis by pyrophosphatase, leading to the release of singly phosphorylated oligosaccharides into the cytosol. We identified that the level of GDP-mannose (Man), which serves as a donor substrate for DLO biosynthesis, is substantially reduced under glucose deprivation. We provide evidence that the selective shutdown of the GDP-Man biosynthetic pathway is sufficient to induce the release of phosphorylated oligosaccharides. These results indicate that glucose-regulated metabolic changes in the GDP-Man biosynthetic pathway cause the biosynthetic arrest of DLOs and facilitate their premature degradation by pyrophosphatase. We propose that this degradation system may avoid abnormal N-glycosylation with premature oligosaccharides under conditions that impair efficient DLO biosynthesis.
Assuntos
Asparagina/metabolismo , Vias Biossintéticas/fisiologia , Dolicóis/metabolismo , Retículo Endoplasmático/metabolismo , Guanosina Difosfato Manose/biossíntese , Oligossacarídeos/biossíntese , Peptídeos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Citosol/metabolismo , Fibroblastos , Glucose/deficiência , Glicosilação , Camundongos , Modelos Biológicos , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Pirofosfatases/metabolismo , Espectrometria de Massas em TandemRESUMO
Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.
Assuntos
Marcação por Isótopo , Espectrometria de Massas/métodos , Nucleotídeos/metabolismo , Polissacarídeos/metabolismo , Animais , Isótopos de Carbono , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Hexosaminas/metabolismo , Ácido Hialurônico/metabolismo , Insulinoma/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Modelos Biológicos , Peso Molecular , Polissacarídeos/biossíntese , Álcoois Açúcares/metabolismo , Fatores de Tempo , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
Glycobiology has contributed tremendously to the discovery and characterization of cancer-related biomarkers containing glycans (i.e., glyco-biomarkers) and a more detailed understanding of cancer biology. It is now recognized that most chronic diseases involve some elements of chronic inflammation; these include cancer, Alzheimer's disease, and metabolic syndrome (including consequential diabetes mellitus and cardiovascular diseases). By extending the knowledge and experience of the glycobiology community regarding cancer biomarker discovery, we should be able to contribute to the discovery of diagnostic/prognostic glyco-biomarkers of other chronic diseases that involve chronic inflammation. Future integration of large-scale "omics"-type data (e.g., genomics, epigenomics, transcriptomics, proteomics, and glycomics) with computational model building, or a systems glycobiology approach, will facilitate such efforts.
Assuntos
Inflamação/metabolismo , Polissacarídeos/metabolismo , Doença de Alzheimer/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Feminino , Glicômica , Humanos , Masculino , Polissacarídeos/química , Doença Pulmonar Obstrutiva Crônica/metabolismo , Biologia de SistemasRESUMO
Glucose is an energy substrate, as well as the primary source of nucleotide sugars, which are utilized as donor substrates in protein glycosylation. Appropriate glycosylation is necessary to maintain the stability of protein, and is also important in the localization and trafficking of proteins. The dysregulation of glycosylation results in the development of a variety of disorders, such as cancer, diabetes mellitus and emphysema. Glycosylation is kinetically regulated by dynamically changing the portfolio of glycosyltransferases, nucleotide sugars, and nucleotide sugar transporters, which together form a part of what is currently referred to as the "Glycan cycle". An excess or a deficiency in the expression of glycosyltransferases has been shown to alter the glycosylation pattern, which subsequently leads to the onset, progression and exacerbation of a number of diseases. Furthermore, alterations in intracellular nucleotide sugar levels can also modulate glycosylation patterns. It is observed that pathological hypoxic microenvironments frequently occur in solid cancers and inflammatory foci. Hypoxic conditions dramatically change gene expression profiles, by activating hypoxia-inducible factor-1, which mediates adaptive cellular responses. Hypoxia-induced glycosyltransferases and nucleotide sugar transporters have been shown to modulate glycosylation patterns that are part of the mechanism associated with cancer metastasis. Hypoxia-inducible factor-1 also induces the expression of glucose transporters and various types of glycolytic enzymes, leading to shifts in glucose metabolic patterns. This fact strongly suggests that hypoxic conditions are an important factor in modulating various nucleotide sugar biosynthetic pathways. This review discusses some of the current thinking of how hypoxia alters glucose metabolic fluxes that can modulate cellular glycosylation patterns and consequently modify cellular functions, particularly from the standpoint of the N-acetylglucosamine cycle, a part of the "Glycan cycle".
RESUMO
Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.