RESUMEN
The hexosamine biosynthetic pathway (HBP) is essential for the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the building block of glycosaminoglycans, thus playing a crucial role in cartilage anabolism. Although O-GlcNAcylation represents a protective regulatory mechanism in cellular processes, it has been associated with degenerative diseases, including osteoarthritis (OA). The present study focuses on HBP-related processes as potential therapeutic targets after cartilage trauma. Human cartilage explants were traumatized and treated with GlcNAc or glucosamine sulfate (GS); PUGNAc, an inhibitor of O-GlcNAcase; or azaserine (AZA), an inhibitor of GFAT-1. After 7 days, cell viability and gene expression analysis of anabolic and catabolic markers, as well as HBP-related enzymes, were performed. Moreover, expression of catabolic enzymes and type II collagen (COL2) biosynthesis were determined. Proteoglycan content was assessed after 14 days. Cartilage trauma led to a dysbalanced expression of different HBP-related enzymes, comparable to the situation in highly degenerated tissue. While GlcNAc and PUGNAc resulted in significant cell protection after trauma, only PUGNAc increased COL2 biosynthesis. Moreover, PUGNAc and both glucosamine derivatives had anti-catabolic effects. In contrast, AZA increased catabolic processes. Overall, "fueling" the HBP by means of glucosamine derivatives or inhibition of deglycosylation turned out as cells and chondroprotectives after cartilage trauma.
Asunto(s)
Vías Biosintéticas/efectos de los fármacos , Enfermedades de los Cartílagos/tratamiento farmacológico , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Glucosamina/farmacología , Hexosaminas/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacología , Biomarcadores/metabolismo , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Enfermedades de los Cartílagos/metabolismo , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo II/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Glicosilación/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analogue of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analogue was activated intracellularly into UDP-4-deoxy-GlcNAc, and HS expression was inhibited up to â¼96% (IC50 = 16 µM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analogue, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors such as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Microinjection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All of these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis, which hampers pro-angiogenic signaling and neo-vessel formation.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Heparitina Sulfato/genética , Neovascularización Patológica/fisiopatología , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Uridina Difosfato N-Acetilglucosamina/farmacología , Animales , Pollos , Regulación hacia Abajo/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/genética , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/metabolismo , Ácido Idurónico/química , Transducción de Señal/efectos de los fármacos , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Pez CebraRESUMEN
We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.
Asunto(s)
N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Uridina Difosfato/síntesis química , Animales , Células CHO , Cricetinae , Difosfatos/química , Activación Enzimática , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Galactosafosfatos/química , Espectroscopía de Resonancia Magnética , N-Acetilglucosaminiltransferasas/química , Proteínas Recombinantes/química , Solventes/química , Tetrazoles/química , Factores de Tiempo , Uridina Difosfato/análogos & derivados , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Glycosyltransferase enzymes play important roles in numerous cellular pathways. Despite their participation in many therapeutically relevant pathways, there is a paucity of information on how to effectively inhibit this class of enzymes. Here we report that UDP-(5F)-GlcNAc acts as a slow-binding, competitive inhibitor of the retaining glycosyltransferase MshA from Corynebacterium glutamicum (K(i) approximately 1.6 muM). The kinetic data are consistent with a single-step inhibition mechanism whose equilibration is slow relative to catalysis. We believe that this is the first slow-onset inhibitor to be reported for the glycosyltransferase family of enzymes. The potent inhibition of the enzyme by the fluoro-substituted substrate is consistent with the involvement of an oxocarbenium transition-state structure, which has been previously proposed for this family of enzymes. Additionally, although several members of the GT-B enzyme family, including MshA, have been shown to undergo a conformational change upon UDP-GlcNAc binding, the kinetic data are inconsistent with a two-step inhibition mechanism. This suggests that there may be other conformations of the enzyme that are useful for the design of inhibitors against the large family of GT-B glycosyltransferase enzymes.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Glicosiltransferasas/metabolismo , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Corynebacterium glutamicum/enzimología , Cinética , Unión Proteica , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Metabolite flux to UDP-GlcNAc and Golgi N-glycan biosynthesis regulates surface residency of glycoprotein receptors and transporters, and thus sensitivities of cells to extracellular cues. Salvage of GlcNAc increases UDP-GlcNAc and branching of N-glycans progressively, but displays an optimum for cell proliferation and bulk endocytosis in mouse NMuMG and human HEK293T epithelial cells. In this report, we measured global changes in gene expression in low and high GlcNAc-supplemented cells. Genes upregulated by high GlcNAc included the EGF and TGF-beta signaling pathways and cell cycle checkpoint, while downregulated genes indicated lower metabolic activity. Genes increased or decreased by high GlcNAc were assessed by transfecting cells with small interfering RNA (siRNA) and measuring effects on three phenotypes: proliferation and bulk endocytosis, and beta1,6GlcNAc-branching of N-glycans. siRNA targeting LGALS3, WBSCR17, PHF3, SDC2 and CTNNAL1 partially reversed the GlcNAc-induced phenotypes, suggesting a role for galectin-3/N-glycans, proteoglycans, O-glycans, and junctional cell adhesion.
Asunto(s)
Perfilación de la Expresión Génica , Genoma Humano/genética , Transducción de Señal/fisiología , Uridina Difosfato N-Acetilglucosamina/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Endocitosis/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
The C-1-phosphonate analogue of UDP-GlcNAc has been synthesized using an alpha-configured C-1-aldehyde as a key intermediate. Addition of the anion of diethyl phosphate to the aldehyde produced the hydroxyphosphonate. The configuration of this key intermediate was determined by X-ray crystallography. Deoxygenation, coupling of the resulting phosphonic acid with UMP and deprotection gave the target molecule as a di-sodium salt. This analogue had no detectable activity as an inhibitor of (OGT).
Asunto(s)
N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Organofosfonatos/síntesis química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Aldehídos , Cristalografía por Rayos X , Estructura Molecular , Organofosfatos , Organofosfonatos/farmacología , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
The inositol 1,4,5-trisphosphate (InsP3) receptor type I (InsP3R-I) is the principle channel for intracellular calcium (Ca2+) release in many cell types, including central neurons. It is regulated by endogenous compounds like Ca2+ and ATP, by protein partners, and by posttranslational modification. We report that the InsP3R-I is modified by O-linked glycosylation of serine or threonine residues with beta-N-acetylglucosamine (O-GlcNAc). The level of O-GlcNAcylation can be altered in vitro by the addition of the enzymes which add [OGT (O-GlcNActransferase)] or remove (O-GlcNAcase) this sugar or by loading cells with UDP-GlcNAc. We monitored the effects of this modification on InsP3R function at the single-channel level and on intracellular Ca2+ transients. Single-channel activity was monitored with InsP3R incorporated into bilayers; Ca2+ signaling was monitored using cells loaded with a Ca2+-sensitive fluorophore. We found that channel activity was decreased by the addition of O-GlcNAc and that this decrease was reversed by removal of the sugar. Similarly, cells loaded with UDP-GlcNAc had an attenuated response to uncaging of InsP3. These results show that O-GlcNAcylation is an important regulator of the InsP3R-I and suggest a mechanism for neuronal dysfunction under conditions in which O-GlcNAc is high, such as diabetes or physiological stress.
Asunto(s)
Glucosamina/análogos & derivados , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Interneuronas/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas , Cerebelo/química , Cerebelo/metabolismo , Glucosamina/química , Glucosamina/metabolismo , Glicosilación , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Interneuronas/efectos de los fármacos , Membrana Dobles de Lípidos/química , Ratones , Microsomas/química , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Rayos Ultravioleta , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacología , beta-N-Acetilhexosaminidasas/químicaRESUMEN
We describe the first synthesis of the C1-phosphonate analog of UDP-GlcNAc, based on a new preparation of the corresponding glycosyl phosphonate. This C-glycosyl analog is shown to be a very weak inhibitor (Ki>10 mM) of fungal chitin synthase, indicating that at least in this case the replacement of the anomeric oxygen with a methylene group is not an innocent substitution.
Asunto(s)
Organofosfonatos/química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Quitina Sintasa/antagonistas & inhibidores , Quitina Sintasa/metabolismo , Glicósidos/química , Modelos Químicos , Estructura Molecular , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. UDP-N-acetylglucosamine 2-epimerase catalyzes the first step of their biosynthesis. Periodate-oxidized UDP-N-acetylglucosamine, namely 2',3'-dialdehydo-UDP-alpha-D-N-acetylglucosamine, was found to be an effective inhibitor of this enzyme, compared with the periodate oxidation products of compounds such as UDP, uridine or methyl riboside. It bound covalently to amino acids in the active site causing an irreversible inhibition. This compound may therefore represent a basis for the synthesis of potent inhibitors of UDP-N-acetylglucosamine 2-epimerase and, as a consequence, of the biosynthesis of sialic acids.
Asunto(s)
Carbohidrato Epimerasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli , Ácidos Siálicos/biosíntesis , Uridina Difosfato N-Acetilglucosamina/farmacología , Animales , Línea Celular , Inhibidores Enzimáticos/química , Estructura Molecular , Oxidación-Reducción , Ácido Peryódico , Spodoptera , Uridina Difosfato N-Acetilglucosamina/químicaRESUMEN
The synthesis and biological evaluation of a new UDP-GlcNAc competitor (I), designed to mimic the transition state of the sugar donor in the enzymatic reaction catalysed by chitin synthetase, is described. Compound (I) was found to competitively inhibit chitin synthetase from Saccharomyces cerevisiae with respect to UDP-GlcNAc, but displayed minimal antifungal activity.
Asunto(s)
Aminoglicósidos , Quitina Sintasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Saccharomyces cerevisiae/enzimología , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Uridina Difosfato N-Acetilglucosamina/farmacología , Antibacterianos/farmacología , Antifúngicos/farmacología , Sitios de Unión , Quitina Sintasa/química , Inhibidores Enzimáticos/química , Cinética , Nucleósidos de Pirimidina/farmacología , Relación Estructura-Actividad , Uridina Difosfato N-Acetilglucosamina/síntesis química , Uridina Difosfato N-Acetilglucosamina/químicaRESUMEN
BACKGROUND: In the endoplasmic reticulum (ER), the stimulation of UDP-glucuronosyltransferase (UGT) by UDP-GlcNAc is based on the interaction of transport across the ER membrane of UDP-GlcUA with UDP-GlcNAc. Intramicrosomal UDP-GlcNAc stimulates influx of UDP-GlcUA and thereby enhances delivery of UDP-GlcUA to the catalytic center of UGT in the ER lumen. AIM: The aim of this study is to investigate whether the interactions between nucleotide sugars for transport across the ER membrane also occur in the Golgi apparatus, and thereby affect UGT activity in Golgi membranes. RESULTS: We found that Golgi membrane preparations display UGT activity which, unlike in ER membranes, is not stimulated by UDP-GlcNAc. Efflux of intravesicular UDP-GlcNAc and UDP-Xyl marginally enhanced uptake of UDP-GlcUA in Golgi vesicles; such trans-stimulation was much more pronounced in the ER. Efflux of intravesicular UDP-GlcNAc was strongly trans-stimulated by cytosolic UDP-GlcUA in ER-derived vesicles but less so in Golgi-derived vesicles. CONCLUSION: The interaction between transport of UDP-GlcUA and transport of UDP-GlcNAc or UDP-Xyl is different in Golgi vesicles compared with ER vesicles. This finding is consistent with the different effects of UDP-GlcNAc on glucuronidation in Golgi and ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Glucuronosiltransferasa/metabolismo , Aparato de Golgi/efectos de los fármacos , Técnicas In Vitro , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Masculino , Ratas , Ratas Wistar , Uridina Difosfato Ácido Glucurónico/farmacología , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacología , Uridina Difosfato Xilosa/metabolismo , Uridina Difosfato Xilosa/farmacologíaRESUMEN
The in vitro complementation assay established by Rothman and co-workers continues to be an important tool to study intra-Golgi transport. In this study, kinetic modeling is used to identify four main parameters that, together, explain the basic features of an assay that is a modification of the original assay. First, the assay signal depends on the ratio of Golgi membranes to transport intermediates in the assay. Secondly, an inactivation rate describes how the activity of transport intermediates decreases over time. Thirdly, the rate at which transport intermediates irreversibly bind to Golgi membranes is measured independently of membrane fusion, thus allowing a quantitative distinction between these two steps. Fourthly, a single rate constant describes the remaining reactions, which result in membrane fusion. This approach of kinetic modeling of experiments is generally applicable to other in vitro assays of cell biological phenomena, permitting quantitative interpretations and an increased resolution of the experiments.
Asunto(s)
Proteínas de Arabidopsis , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana , Proteínas de Plantas , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Glicosilación , Hexosaminidasas/metabolismo , Cinética , Hígado/metabolismo , Modelos Biológicos , Transporte de Proteínas , Ratas , Factores de Tiempo , Factores de Transcripción/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
For the enzymatic transfer of galactose, N-acetylglucosamine, and N-acetylgalactosamine, UDP-Gal (1), UDP-GlcNAc (2), and UDP-GalNAc (3) are employed, and UDP serves as a feedback inhibitor. In this paper the synthesis of the novel UDP-sugar analogues 4, 5, and 6 as potential transferase inhibitors is described. Compounds 4-6 feature C-glycosidic hydroxymethylene linkages between the sugar and nucleoside moieties in contrast to the anomeric oxygens in the natural derivatives 1-3.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Transferasas/antagonistas & inhibidores , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato N-Acetilgalactosamina/síntesis química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética , Imitación Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato N-Acetilgalactosamina/química , Uridina Difosfato N-Acetilgalactosamina/farmacología , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.
Asunto(s)
Acetilglucosamina/análogos & derivados , Quitina/química , Oligosacáridos/biosíntesis , Rhizobiaceae/química , Pez Cebra/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Conformación de Carbohidratos , Quitina/antagonistas & inhibidores , Quitina/biosíntesis , Embrión no Mamífero/metabolismo , Escherichia coli/química , Escherichia coli/genética , Glucosamina/análogos & derivados , Glucosamina/química , Glucosamina/metabolismo , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Oligosacáridos/antagonistas & inhibidores , Oligosacáridos/química , Rhizobiaceae/genética , Rhizobiaceae/metabolismo , Especificidad por Sustrato , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Uridina Difosfato N-Acetilglucosamina/farmacología , Pez Cebra/embriologíaRESUMEN
Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited Km(Gln) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with kcat = 1150 min-1. The isoelectric point of 4.6 +/- 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with nH = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1. 21 +/- 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast --> mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Animales , Northern Blotting , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Focalización Isoeléctrica , Cinética , Peso Molecular , Conformación Proteica , Proteínas Recombinantes , Mapeo Restrictivo , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Toxoplasma gondii is an obligate intracellular parasite of the phylum apicomplexa and a common and often life-threatening opportunistic infection associated with AIDS. A family of parasite-specific glycosylphosphatidylinositols containing a novel glucosylated side chain has been shown to be highly immunogenic in humans (Striepen et al. (1997) J. Mol. Biol. 266, 797-813). In contrast to trypanosomes in T. gondii side chain modification takes place before addition to protein in the endoplasmic reticulum. The biosynthesis of these modifications was studied in an in vitro system prepared from hypotonically lysed T. gondii parasites. Radiolabeled glucose-containing glycosylphosphatidylinositol precursors were synthesized by T. gondii membrane preparations upon incubation with uridine diphosphate-[3H]glucose. Synthesis of glucosylated glycolipids took place only in the presence of exogenous uridine diphosphate-glucose and was stimulated by unlabeled uridine diphosphate-glucose in a dose-dependent manner. In contrast to glycosylphosphatidylinositol mannosylation, glucosylation was shown to be insensitive to amphomycin treatment. In addition, the glucose analogue 2-deoxy-D-glucose was used to trace the glycosylphosphatidylinositol glucosylation pathway. Detailed analysis of glycolipids synthesized in vitro in the presence of UDP and GDP derivatives of D-glucose and 2-deoxy-D-glucose ruled out an involvement of dolichol phosphate-glucose and demonstrates direct transfer of glucose from uridine diphosphate-glucose.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Uridina Difosfato Glucosa/análogos & derivados , Uridina Difosfato Glucosa/metabolismo , Animales , Antibacterianos/farmacología , Bovinos , Sistema Libre de Células , Glucosa/metabolismo , Glucolípidos/química , Glucolípidos/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/química , Azúcares de Guanosina Difosfato/química , Humanos , Lipopéptidos , Oligopéptidos/farmacología , Polisacáridos/química , Polisacáridos/metabolismo , Toxoplasma , Uridina Difosfato Glucosa/química , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
The enzyme UDP-N-acetylglucosamine (UDP-GlcNAc) enolpyruvyltransferase (MurA), the target of the antibiotic fosfomycin, was investigated by small-angle X-ray scattering (SAXS) and fluorescence spectroscopy to detect conformational changes that had been proposed on the basis of the crystal structure of unliganded and liganded MurA. The SAXS data indicate that binding of UDP-GlcNAc to free enzyme results in substantial conformational changes, which can be interpreted as the transition from an open to a closed form. Fosfomycin did not affect the structure of free enzyme or sugar-nucleotide-bound MurA. Phosphoenolpyruvate (pyruvate-P) appeared to induce a structural change upon addition to free enzyme, which differed from that observed upon binding of UDP-GlcNAc. Fluorescence experiments were performed using the hydrophobic fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS). The fluorescence quenching of MurA/ANS solutions upon addition of UDP-GlcNAc or pyruvate-P was concentration dependent in a saturating manner, yielding apparent dissociation constants of K(d(UDP-GlCNAc)) = 59 microM and K(d(pyruvate-P)) = 240 microM. The results suggest that binding of substrates does not exclusively follow an ordered mechanism with UDP-GlcNAc binding first, although binding of UDP-GlcNAc to free enzyme is preferred and possibly influenced by pyruvate-P. The reaction thus appears to follow an induced-fit mechanism, in which the binding site for fosfomycin, and presumably also for pyruvate-P, is created by the interaction of free enzyme with the sugar nucleotide. The methods described here provide a tool for the characterization of site-directed mutants of MurA and the interaction of this enzyme with potential inhibitors.
Asunto(s)
Transferasas Alquil y Aril/química , Proteínas Bacterianas/química , Conformación Proteica , Cristalografía por Rayos X , Enterobacter cloacae/enzimología , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
To evaluate whether a temporary hepatic insufficiency may affect intestinal glucuronidation, we determined UDP-glucuronosyltransferase activity towards bilirubin and p-nitrophenol in rat jejunum and liver after partial hepatectomy. Enzyme assays were performed in native, and in UDP-N-acetylglucosamine- or palmitoyl lysophosphatidylcholine-activated microsomes at different times post-hepatectomy. Content of enzyme was analyzed by Western blot. Microsomal cholesterol/phospholipid ratio, phospholipid and total fatty acid classes were also determined to evaluate the possible influence on enzyme activity. The results show that while hepatic microsomes exhibited no change in UDP-glucuronosyltransferase activity (for both substrates) with respect to shams at any time of study, intestinal activities increased significantly 48 h after surgery, returning to sham values 96-h post-hepatectomy. Western blotting confirmed the increase (about 50% for both substrates 48-h post-hepatectomy) in intestinal UDP-glucuronosyltransferase activity. No variations were observed in hepatic and intestinal microsomal lipid composition in agreement with the absence of modification in the percent of activation by palmitoyl lysophosphatidylcholine. In conclusion, jejunum but not liver, was able to produce a compensatory increase in conjugation capacity during a transitory loss of hepatic mass. The phenomenon is associated to a modification in the amount of UDP-glucuronosyltransferase, rather than to changes in the characteristics of the enzyme environment.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Hepatectomía , Mucosa Intestinal/enzimología , Animales , Bilirrubina/sangre , Activación Enzimática/efectos de los fármacos , Glucuronosiltransferasa/fisiología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Yeyuno , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Ratas , Ratas Wistar , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5'-phosphate. The enzyme is extremely tolerant to changes at the 3'-position, and the presence of 3'-F actually increases binding of the NMP to the enzyme. At the 2'-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Aparato de Golgi/ultraestructura , Membranas Intracelulares/metabolismo , Hígado/ultraestructura , Nucleótidos/farmacología , Uridina Difosfato N-Acetilglucosamina/metabolismo , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , Glicosilación , Cinética , Nucleótidos/química , Fosfatos/química , Fosfatos/farmacología , Conejos , Ribosa/química , Relación Estructura-Actividad , Uridina Difosfato N-Acetilglucosamina/farmacología , Azúcares de Uridina Difosfato/metabolismo , Azúcares de Uridina Difosfato/farmacologíaRESUMEN
To clarify the character of the anticoccidial activity of diclazuril a series of battery trials was conducted. Diclazuril showed excellent anticoccidial activity in the infection of chickens with Eimeria tenella, E. necatrix or E. acervulina at the feeding level of 0.1 ppm. When diclazuril was administered in combination with a nucleic acid precursor, uracil, uridine, orotate or orotidine, the reduction of the activity of diclazuril to the infections induced by above species was not observed. While, bloody droppings with severe cecal lesions were resulted, when diclazuril was administered in combination with uridine 5(1)-diphosphoglucose (UDPG) or its N-acetyl amine (UDPGNAC) to chickens infected with E. tenella. While, body weight gain of the birds and oocyst output was not affected by these combination-treatment. Results demonstrated that the antagonistic effect of UDPG and UDPGNAC to diclazuril was partial. The possibility of the cross resistance between diclazuril and 6-azauracil (AzU) in E. tenella was investigated using two populations induced resistance to AzU or diclazuril. The results demonstrated that the cross resistance does not exist between AzU and diclazuril, indicating that the mode of action of each drug is different.