Dataset Information

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

ABSTRACT: The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

SUBMITTER: Adler LN

PROVIDER: S-EPMC3122210 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

Adler Lital N LN Gomez Tara A TA Clarke Steven G SG Linster Carole L CL

The Journal of biological chemistry 20110420 24

The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP ...[more]

PMID: 21507950

Similar Datasets

Project description:Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.

Dataset Information

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

Publications

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets