RESUMO
The O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a critical post-translational modification and closely linked to various physiological and pathological conditions. The O-GlcNAc transferase (OGT) functions as the only glycosyltransferase of O-GlcNAc glycosylation by transferring GlcNAc from UDP-GlcNAc to serine or threonine residues on protein substrates. The interaction mode of UDP-GlcNAc against OGT has been preliminarily revealed by the crystal structures, yet an atomic-level comprehension for the conformational dynamics of the recognition process remains elusive. Here, we construct the Markov state model based on extensive all-atom molecular dynamics (MD) simulations with an aggregated simulation time of â¼9 µs, and reveal that the UDP-GlcNAc recognition process by OGT encompasses four key metastable states, occurring within an estimated timescale of â¼10 µs. During UDP-GlcNAc recognition process, we find the pyrophosphate moiety (P2O52-) initially anchors to the active pocket via salt bridge and hydrogen bonds, facilitating subsequent binding of the uridine and GlcNAc moieties. Furthermore, the functional roles of K842 involved in the salt bridge with P2O52- were evaluated through extra mutant MD simulations. Overall, our study provides valuable insights into the UDP-GlcNAc recognition mechanism by OGT, which could further aid in mechanistic studies of O-GlcNAc glycosylation and drug development targeting on OGT.
Assuntos
Simulação de Dinâmica Molecular , N-Acetilglucosaminiltransferases , Processamento de Proteína Pós-Traducional , Glicosilação , Difosfato de Uridina/metabolismo , Acetilglucosamina/metabolismoRESUMO
AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin â ¢, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin â ¢-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin â ¢ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin â ¢. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin â ¢ semi-synthesis.
Assuntos
Acetilglucosamina/metabolismo , Alcaloides/biossíntese , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Alcaloides/economia , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/economia , Biocatálise , Paclitaxel/biossíntese , Paclitaxel/química , Paclitaxel/economia , Especificidade por Substrato , Taxoides/economiaRESUMO
Human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC CMs) may be used in regenerative medicine for individualized tissue transplants in the future. For application in patients, the generated CMs have to be highly pure and well characterized. In order to overcome the prevalent scarcity of CM-specific markers, we quantitatively assessed cell-surface-exposed sialo-glycoproteins and N-glycans of hiPSCs, CM progenitors, and CMs. Applying a combination of metabolic labeling and specific sialo-glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation. Among them we identified a number of novel, putative biomarkers for hiPSC CMs. Analysis of the N-glycome by capillary gel electrophoresis revealed three novel structures comprising ß1,3-linked galactose, α2,6-linked sialic acid and complex fucosylation; these were highly specific for hiPSCs. Bisecting GlcNAc structures strongly increased during differentiation, and we propose that they are characteristic of early, immature CMs.
Assuntos
Membrana Celular/química , Glicômica/métodos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , Polissacarídeos/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sequência de Carboidratos , Diferenciação Celular , Membrana Celular/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Fucose/química , Fucose/metabolismo , Galactose/química , Galactose/metabolismo , Gastrinas/genética , Gastrinas/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Polissacarídeos/metabolismo , Receptor EphA7/genética , Receptor EphA7/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Coloração e Rotulagem/métodosRESUMO
AIMS/HYPOTHESIS: O-GlcNAcylation plays a role as a metabolic sensor regulating cellular signalling, transcription and metabolism. Transcription factors and signalling pathways related to metabolism are modulated by N-acetyl-glucosamine (O-GlcNAc) modification. Aberrant regulation of O-GlcNAcylation is closely linked to insulin resistance, type 2 diabetes and obesity. Current evidence shows that increased O-GlcNAcylation negatively regulates insulin signalling, which is associated with insulin resistance and type 2 diabetes. Here, we aimed to evaluate the effects of Oga (also known as Mgea5) haploinsufficiency, which causes hyper-O-GlcNAcylation, on metabolism. METHODS: We examined whether Oga(+/-) mice developed insulin resistance. Metabolic variables were determined including body weight, glucose and insulin tolerance, metabolic rate and thermogenesis. RESULTS: Oga deficiency does not affect insulin signalling even at hyper-O-GlcNAc levels. Oga(+/-) mice are lean with reduced fat mass and improved glucose tolerance. Furthermore, Oga(+/-) mice resist high-fat diet-induced obesity with ameliorated hepatic steatosis and improved glucose metabolism. Oga haploinsufficiency potentiates energy expenditure through the enhancement of brown adipocyte differentiation from the stromal vascular fraction of subcutaneous white adipose tissue (WAT). CONCLUSIONS/INTERPRETATION: Our observations suggest that O-GlcNAcase (OGA) is essential for energy metabolism via regulation of the thermogenic WAT program.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Metabolismo Energético/genética , Obesidade/genética , beta-N-Acetil-Hexosaminidases/genética , Acetilglucosamina/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Marrons/patologia , Animais , Glicemia/metabolismo , Peso Corporal/genética , Diferenciação Celular , Diabetes Mellitus Tipo 2/genética , Intolerância à Glucose/genética , Resistência à Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Termogênese/genéticaRESUMO
We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.
Assuntos
Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Escherichia coli/enzimologia , Microbiologia Industrial/métodos , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Escherichia coli/genética , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Deleção de Genes , Melhoramento Genético/métodos , Lactose/análogos & derivados , Lactose/metabolismo , Mutagênese Insercional , N-Acilneuraminato Citidililtransferase/genética , N-Acilneuraminato Citidililtransferase/metabolismo , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Oligossacarídeos/biossíntese , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Transformação BacterianaRESUMO
We have adapted two methods to evaluate the beta-hexosaminidase (HEX) enzymatic activity in cultured cells, based on the use of (i) the fluorogenic substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2- deoxy-beta-D-glucopyranoside (MU-GlcNAc-6-SO4) and (ii) the naphthol AS-BI-N-acetyl-beta-D-glucosaminide and hexazotized pararosaniline. We demonstrate that both methods could be used for the HEX isoenzymes by comparing wild-type and mutant human fibroblast cell lines, deficient for either an alpha or beta subunit from Tay-Sachs and Sandhoff patients. This in situ cytochemical assessment of HEX activity offers a rapid evaluation to study the expression of this enzyme in a heterogeneous cell population such as in gene transfer experiments.
Assuntos
Isoenzimas/metabolismo , Doença de Sandhoff/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/metabolismo , Células Cultivadas , Fibroblastos/enzimologia , Glucuronatos/metabolismo , Histocitoquímica , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Isoenzimas/genética , Mutação , Naftóis , Espectrometria de Fluorescência , Doença de Tay-Sachs/enzimologia , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
The Metropolis technique of conformation searching is combined with rapid energy evaluation using molecular affinity potentials to give an efficient procedure for docking substrates to macromolecules of known structure. The procedure works well on a number of crystallographic test systems, functionally reproducing the observed binding modes of several substrates.