Nucleoside Diphosphosugar Research Articles

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.

Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme.

Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom) SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate. Stress-induced SlSGT2 expression largely parallels that of SlSGT4. On the contrary, SlSGT1 and SlSGT3 expression remains almost unaltered under the tested stress conditions. Overall, this study contributes to broaden the current knowledge on plant SGTs and provides support to the view that tomato SGTs play overlapping but not completely redundant biological functions involved in mediating developmental and stress responses.

Metal ion-promoted cleavage of nucleoside diphosphosugars: a model for reactions of phosphodiester bonds in carbohydrates.

Cleavage of five different nucleoside diphosphosugars has been studied in the presence of Cu(2+) and Zn(2+) complexes. The results show that metal ion catalysts promote the cleavage via intramolecular transesterification whenever a neighbouring HO group can adopt a cis-orientation with respect to the phosphate. The HO group attacks the phosphate and two monophosphate products are formed. If such a nucleophile is not available, Cu(2+) complexes are able to promote a nucleophilic attack of an external nucleophile, e.g. a water molecule or metal ion coordinated HO ligand, on phosphate. With the Zn(2+) complex, this was not observed.

Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase

Free ADP-ribose has a reducing ribose moiety and it is hazardous due to its nonenzymic reactivity toward protein side chains. ADP-ribose hydrolases are putative protective agents to avoid the intracellular accumulation of ADP-ribose. In mammalian sources, two types of enzymes with ADP-ribose hydrolase activity are known: (i) highly specific ADP-ribose pyrophosphatases, which in a Mg 2+-dependent fashion hydrolyse only ADP-ribose and the nonphysiological analogue IDP-ribose, and (ii) less specific nucleoside diphosphosugar or diphosphoalcohol (NDP-X) pyrophosphatases, which besides A(I)DP-ribose hydrolyse also some nonreducing NDP-X substrates. So far, of these two enzyme types only the less specific one has been reported in human sources: an ADP-sugar pyrophosphatase purified from erythrocytes or expressed from cDNA clones. Here we report that human placenta extracts contain two ADP-ribose hydrolases, which were characterised after a near 1000-fold purification. One is an ADP-sugar pyrophosphatase: it hydrolysed ADP-ribose, ADP-glucose and ADP-mannose, but not e.g. UDP-glucose, at similar rates. It resembles the erythrocyte and recombinant enzyme(s), but showed a 5–20-fold lower K m for ADP-ribose (7 μM). The other enzyme is a highly specific ADP-ribose pyrophosphatase (the first of this kind to be reported in humans): it hydrolysed only ADP-ribose and IDP-ribose at similar rates, with a very low, 0.4 μM K m for the former. This is a major candidate to control the accumulation of free ADP-ribose in humans. It remains to be seen whether it belongs to the ‘nudix’ protein family, which includes several ADP-ribose hydrolases and other ‘housecleaning’ enzymes (M.J. Bessman, D.N. Frick, S.F. O’Handley, J. Biol. Chem. 271 (1996) 25059-25062).

Metabolite mapping of human filarial parasite, Brugia Malayi with nuclear magnetic resonance

Metabolite mapping of human filarial parasite, Brugia malayi was carried out in vitro as well as in situ in host Mastomys coucha by 31P nuclear magnetic resonance (NMR) spectroscopy. Detection of parasites by visualizing contrast spots due to pathologic changes was observed by 1H magnetic resonance imaging (MRI). Major metabolites of adult B. malayi observed by 31P-NMR spectroscopy were of sugar phosphates (SP), phosphomonoesters (PME), glycerophosphoryl-ethanolamine (GPE), -choline (GPC), phosphoenolpyruvate (PEP), inorganic phosphate (Pi), nucleoside diphosphosugar and nucleotides-mono, -di and -tri phosphates. PEP and GPC were present in high concentration; PEP being the major energy reservoir and GPC the major phospholipid in this species of filaria. The 31P NMR spectra of testis of mastomys, showed seven major peaks of SP, PME, phosphocreatine (PCr), phosphodiesters (PDE), Pi, and nucleotides di- and tri-phosphates. The 31P-NMR spectra of testis of B. malayi infected animal also consisted of seven major peaks with significant decrease in the SP and PME peak showing changes in the carbohydrate and lipid metabolism of filaria infected testis. Thus, in vivo 31P MRS provided a non-invasive assessment of tissue bioenergetics and phospholipid metabolism.

Rat liver nucleoside diphosphosugar or diphosphoalcohol pyrophosphatases different from nucleotide pyrophosphatase or phosphodiesterase I: substrate specificities of Mg 2+- and/or Mn 2+-dependent hydrolases acting on ADP-ribose

Three rat liver nucleoside(5′) diphosphosugar (NDP-sugar) or nucleoside(5′) diphosphoalcohol pyrophosphatases are described: two were previously identified in experiments measuring Mg 2+-dependent ADP-ribose pyrophosphatase activity (Miró et al. (1989) FEBS Lett. 244, 123–126), and the other is a new, Mn 2+-dependent ADP-ribose pyrophosphatase. They are resolved by ion-exchange chromatography, and differ by their substrate and cation specificities, K M values for ADP-ribose, pH-activity profiles, molecular weights and isoelectric points. The enzymes were tested for activity towards: reducing (ADP-ribose, IDP-ribose) and non-reducing NDP-sugars (ADP-glucose, ADP-mannose, GDP-mannose, UDP-mannose, UDP-glucose, UDP-xylose, CDP-glucose), CDP-alcohols (CDP-glycerol, CDP-ethanolamine, CDP-choline), dinucleotides (diadenosine pyrophosphate, NADH, NAD +, FAD), nucleoside(5′) mono- and diphosphates (AMP, CMP, GMP, ADP, CDP) and dTMP p-nitrophenyl ester. Since the enzymes have not been purified to homogeneity, more than three pyrophosphatases may be present, but the co-purification of activities, thermal co-inactivation, and inhibition experiments give support to: (i) an ADP-ribose pyrophosphatase highly specific for ADP(IDP)-ribose in the presence of Mg 2+, but active also on non-reducing ADP-hexoses and dinucleotides (not on NAD +) when Mg 2+ was replaced with Mn 2+; (ii) a Mn 2+-dependent pyrophosphatase active on ADP(IDP)-ribose, dinucleotides and CDP-alcohols; (iii) a rather unspecific pyrophosphatase that, with Mg 2+, was active on AMP(IMP)-containing NDP-sugars and dinucleotides (not on NAD +), and with Mn 2+, was also active on non-adenine NDP-sugars and CDP-alcohols. The enzymes differ from nucleotide pyrophosphatase/phosphodiesterase-I (NPPase/PDEaseI) by their substrate specificities and by their cytosolic location and solubility in the absence of detergents. Although NPPase/PDEaseI is much more active in rat liver, its known location in the non-cytoplasmic sides of plasma and endoplasmic reticulum membranes, together with the known cytoplasmic synthesis of NDP-sugars and CDP-alcohols, permit the speculation that the pyrophosphatases studied in this work may have a cellular role.

Metabolic Compatibility of Abattoir and Human Corneas: An Ex Vivo 31P Nuclear Magnetic Resonance Spectroscopic Study of Intact Tissues

This study establishes the metabolic similarities and differences of intact abattoir corneas and the human cornea ex vivo, using 31P nuclear magnetic resonance spectroscopy. Phosphorylated sugars (SP), inorganic orthophosphate (Pi), phosphodiesters, ATP, ADP, and the nucleoside diphosphosugars and dinucleotides (NS&DN) were quantitated. The intracorneal pH also was determined. In addition, metabolic indices ATP/Pi, ATP/ADP, ATP/(ADP + Pi), SP/Pi, the phosphorylation potential, and the 31P energy modulus were calculated. Significant differences were observed between the abattoir and human corneas in phosphorylated sugars, NS&DN, and intracorneal pH, as well as in the indices ATP/ADP and SP/Pi. The overall energy status as measured by the 31P energy modulus was significantly higher in the bovine cornea when compared to the porcine and human corneas, suggestive of a very high energy reserve in the bovine cornea.

Phosphatic intermediate metabolites of the porcine ocular tunica fibrosa

This study compares porcine scleral and corneal phosphorylated intermediate metabolites determined using 31P NMR. These tissues comprise the tunica fibrosa (outer coat) of the eye. Since the sclera does not possess epithelia as does the cornea, comparative analysis of these tissues included examination of the cornea with and without its epithelia. The phosphorylated intermediates detected include: dihydroxyacetone phosphate, hexose 6-phosphates, alpha-glycerol phosphate, beta-glycerol phosphate, ethanolamine and choline phosphates, nucleoside mono-, di-, and tri-phosphates, inorganic orthophosphate, glycerol 3-phosphoryl-ethanolamine and -choline, phosphoglycans, phosphocreatine, nucleoside diphosphosugars, dinucleotides, and four uncharacterized (unknown) signals. Metabolic indices, comprised of individual or grouped metabolites, were calculated to further compare and contrast metabolites and to provide more pathway specific metabolic interrelations for phosphorylated metabolites. Significant differences exist between the corneal stroma and the sclera in 16 of the 22 phosphorylated metabolites determined, whereas differences exist between the whole cornea and the sclera in six of the 22 phosphorylated metabolites. Considering all metabolite levels and metabolic indices in aggregate, the sclera is most similar to the whole cornea and least similar to the corneal stroma.

Interspecies analysis of corneal phosphate metabolites

Phosphate metabolite profiles of corneas were determined for human, baboon, monkey, cat, guinea-pig, pig, dog, rabbit, and cow using 31P-nuclear magnetic resonance (NMR) spectroscopy. Metabolites quantitatively measured included the low-energy metabolites: hexose 6-phosphates, alpha-glycerophosphate, unassigned phosphorus compound(s) at 4.26-3.91 delta, ethanolamine phosphate, nucleoside monophosphates, choline phosphate, inorganic orthophosphate, phosphodiesters (glycerol 3-phosphoethanolamine and glycerol 3-phosphocholine), and the high-energy metabolites: phosphocreatine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), nucleoside diphosphosugars and dinucleotides. The number of significant differences in metabolites among the species studied, relative to humans, increased in the following order: baboon less than monkey approximately pig approximately cow less than cat approximately guinea-pig less than rabbit less than dog. The 31P energy modulus (human, 1.3 +/- 0.20 S.D.). ATP:Pi ratio (human, 0.5 +/- 0.05 S.D.). ATP:ADP ratio (human, 0.38 +/- 0.34 S.D.). ATP:ADP plus Pi ratio (human, 0.43 +/- 0.04 S.D.) and SP:Pi ratio (human, 0.37 +/- 0.04 S.D.) were determined for each species. All these ratios are indicators of corneal metabolic status, and their values suggest that, of the common laboratory research animals examined, the baboon and monkey are the most like man, and the rabbit and cow the least like man.

Effects of the slow calcium-channel blocker verapamil on phosphatic metabolism of crystalline lens

The effects of calcium-channel blockade on phosphate metabolism in the rabbit lens ex vivo was studied using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Rates-of-change of the intralenticular pH and the following phosphatic metabolites in the lens were determined: ATP, ADP, inorganic orthophosphate, sugar phosphates, nucleoside diphosphosugars, phosphodiesters, dinucleotides, and an uncharacterized resonance peak at 6 delta in the 31P spectrum. Incubation in 20, 200 or 2000 micrograms/ml verapamil led to the same qualitative changes in the lens's metabolic profile at each different concentration; only the rates of metabolite change were altered significantly by verapamil concentration. Compared with the control, the entire orthophosphate resonance band increased relative to the signals of ATP, and the alpha-glycerophosphate and nucleoside monophosphate resonances increased in relation to inorganic orthophosphate, and these two resonance groups shifted relative to each other so that they coalesced, indicating that the two groups of signals arose from compounds in relatively different pH compartments. In addition to altering metabolic rates, verapamil reduced the intralenticular pH of the sugar phosphate and inorganic orthophosphate pools with respect to those containing the nucleoside monophosphates; the pH-change difference was about one order of magnitude smaller for the mononucleotide pool. Despite the metabolic alterations and changes in pH during verapamil incubation, lens clarity was unchanged.

Phosphorus-31 NMR analysis of dynamic energy metabolism in intact crystalline lens treated with ouabain: phosphorylated metabolites.

Changes in phosphatic metabolites and intralenticular pH of the intact crystalline rabbit lens induced by ouabain (10(-3), 10(-4), 10(-5) M) were measured during time-course incubations using phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) in order to study the metabolic manifestations of this steroid on lens glycolytic activity. Significant alterations in the glycolytic intermediates of the crystalline lens only occurred with ouabain at the concentration of 10(-3) M. A significant time-dependent increase in alpha-glycerophosphate, an unidentified pentose phosphate, the nucleoside diphosphosugars (uridine diphosphoglucose and uridine diphosphomannose), inorganic orthophosphate, and inosine monophosphate, and decline in glucose 6-phosphate and ATP were detected in 10(-3) M ouabain-treated lenses. The uridine diphosphosugars exhibited a maximum at approximately the time when the lens ATP content was 50% of control levels (12.5 h). A progressive decrease in intralenticular pH indicative of hydrogen-ion-pump damage was detected concomitant with the changes in lens metabolite levels. Lenses maintained transparency throughout the time-course. Since metabolite manifestations effecting the intermediates of glycolysis occur in lens tissue with its high glycolytic and low oxidative activity, we hypothesize the effects of 10(-3) M ouabain may represent metabolic actions of ouabain which are independent of its effects on tissue oxygen consumption as is presumed from studies involving tissues more dependent on oxidative metabolism.

Organophosphate metabolites of the human and rabbit crystalline lens: a phosphorus-31 nuclear magnetic resonance spectroscopic analysis

The concentrations of the major and minor organophosphate metabolites of the rabbit and human crystalline lens were quantitated from perchloric acid extracts analyzed by phosphorus-31 nuclear magnetic resonance spectroscopy. The concentrations of adenosine diphosphate, phosphocreatine, inorganic orthophosphate and the nucleoside diphosphosugars were greater in the human lens samples when compared with the rabbit lens profile. A 10-fold greater phosphocretine content of the human lens was the most remarkable species difference. The adenosine triphosphate and the glycerol derivative, glycerol 3-phosphorylcholine, were enriched in the rabbit lens relative to the human. A prominent resonance signal at 6 p.p.m. corresonding to an as yet, unidentified phosphorus-containing molecule and two other less prominent phosphorus resonances of unidentified origin at 17·9 and 10·7 p.p.m., detected in the rabbit lens were absent from the human lens profile. In addition, the α-glycerophosphate content of the human lens was considerably less than that of the rabbit lens. These profile data document similarities and differences in the human versus the rabbit lens metabolites. The magnitude and diversity of these differences in lens metabolite concentrations emphasize the need for cautious rabbit to human extrapolation of experimental findings.

Purification and properties of a nucleoside diphosphosugar : NAD + 2-hexosyl oxidoreductase

Penicillium charlesii contains a nucleoside diphosphosugar : NAD 2-hexosyl oxidoreductase that oxidizes UDPgalactose (UDP-Gal p), ADPribose [1,2] and UDPglucose (UDP-Glc). Dithiothreitol, NAD and 0.25 M NaCl but not nucleoside diphosphates stabilize the enzyme activity. The enzyme was purified and separated on polyacrylamide disc gels by electrophoresis into one major and eight minor bands of protein. Oxidoreductase activity was located in the major and three of the minor bands of protein. Each of these proteins catalyze the oxidation of UDPGal p, ADPribose and UDP-Glc.

Transfer of Sugars from Nucleoside Diphosphosugar Compounds to Endogenous and Synthetic Dolichyl Phosphate in Human Lymphocytes

Abstract Whole cell homogenate or membranes from human lymphocytes catalyzed the transfer of sugars from nucleoside diphosphosugar compounds to lipid and complex glycan. Transfer from GDP-mannose, UDP-glucose, or UDP-acetylglucosamine to lipid was stimulated by synthetic dolichyl phosphate (a new synthesis of which is described). No lipid was produced from GDP-fucose, UDP-galactose, or CMP-sialic acid, but complex glycan was labeled in all cases. The lipid formed from GDP-mannose and either endogenous acceptor or synthetic dolichyl phosphate chromatographed in the same position as synthetic dolichyl phosphomannose in three solvent systems, and was further characterized as dolichyl phosphomannose. Products formed by stimulation with C55-ficaprenyl phosphate, C45-solanesyl phosphate, or C15-farnesyl phosphate could be distinguished chromatographically from dolichyl phosphomannose. Three lipid products were formed from UDP-glucose. The formation of one of these which chromatographed near dolichyl phosphomannose was stimulated by dolichyl phosphate. Three lipid products were formed in small amounts from UDP-acetylglucosamine. Formation of all three was stimulated by dolichyl phosphate.