A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase.

We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.     

Evidence of partial localization in Golgi apparatus of UDP-glucose collagen glucosyltransferase from chick embryo liver

1. Collagens are the most important components of the connective tissue. 2. Collagen synthesis involves greater than 12 different enzymes whereas three enzymatic systems are involved in the ordered degradation. 3. Some enzymes are found in the rough endoplasmic reticulum (RER). The subcellular localization of disulfur isomerase, alpha D-glucosidase, proteases, galactosyltransferases and glucosyltransferases specific to collagen is unknown. 4. After having determined the best subcellular fractionation conditions for the chick embryo liver, we demonstrate that the galactosylhydroxylysyl glucosyltransferase specific to collagen is located in the RER and in the Golgi apparatus.     

Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and right-side-out Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process.Abbreviations Chaps 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate - Mops 4-morpholinepropanesulfonic acid - PC phosphatidylcholine - NEM N-ethylmaleimide - CPDS carboxypyridine disulfide (dithio-6,6-dinicotinic acid) - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - DTP dithiodipyridine - p-HMB para-hydroxymercuribenzoate - DTT dithiothreitol - BAL British anti-Lewisite (dimercaptopropanol) - Zw 3–14 Zwittergent 3–14     

Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast. They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.     

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.     

DNA damage induces down-regulation of UDP-glucose ceramide glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells

DNA damaging agents typically induce an apoptotic cascade in which p53 plays a central role. However, absence of a p53-mediated response does not necessarily abrogate programmed cell death, due to the existence of p53-independent apoptotic pathways, such as those mediated by the pro-apoptotic molecule ceramide. We compared ceramide levels before and after DNA damage in human osteosarcoma (U2OS) and colon cancer (HCT116) cells that were either expressing or deficient in p53. When treated with mitomycin C, p53-deficient cells, but not p53-expressing cells, showed a marked increase in ceramide levels. Microarray analysis of genes involved in ceramide metabolism identified acid ceramidase (ASAH1, up-regulated), ceramide glucosyltransferase (UGCG, down-regulated), and galactosylceramidase (GALC, up-regulated) as the three genes most affected. Experiments employing pharmacological and siRNA agents revealed that inhibition of UGCG is sufficient to increase ceramide levels and induce cell death. When inhibition of UGCG and treatment with mitomycin C were combined, p53-deficient, but not p53-expressing cells, showed a significant increase in cell death, suggesting that the regulation of sphingolipid metabolism could be used to sensitize cells to chemotherapeutic drugs.     

Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato

A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato. The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms. The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP-glucosyltransferases. The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato. SGT mRNA and protein accumulated in tubers in response to wounding. The time course for SGT mRNA accumulation paralleled that of 3-hydroxy-3-methylglutaryl-coenzymeA isoform 1 ( hmg1 ) mRNA. Steady-state SGT mRNA levels also increased transiently upon wounding of leaves.     

Isolation and characterization of UDP-glucose dolichyl-phosphate glucosyltransferase from human liver

The enzyme UDP-glucose dolichyl-phosphate glucosyltransferase has been purified to near homogeneity from human liver microsomes. A 1100-fold enrichment over starting microsomal membranes was achieved by selective solubilization followed by anion- and cation-exchange chromatography, 5-HgUDP-thiopropyl-Sepharose affinity chromatography, butylagarose chromatography and hydroxyapatite chromatography. The glucosyltransferase was shown to be separated from other dolichyl-phosphate-dependent glycosyltransferases catalyzing the formation of dolichyl diphospho-N-acetylglucosamine and dolichyl phosphomannose. Sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis of the purified enzyme under reducing conditions revealed a protein band of Mr 36,000. Protection of the solubilized enzyme against rapid inactivation was achieved by its competitive inhibitor uridine. The purified glucosyltransferase activity exhibited a specific requirement for the presence of phospholipids. Phosphatidylethanolamine was the most effective activator of enzyme activity.     

Solubilization of UDPglucose-ceramide glucosyltransferase from the Golgi apparatus

The choice of a suitable detergent for solubilization of UDPglucose-ceramide glucosyltransferase from Golgi membranes has been investigated. Among the various classes of detergent, CHAPS, a zwitterionic detergent, was used as it produced a substantial activation of the enzyme activity. 30% of the enzyme activity and 50% of proteins were solubilized in the first attempts. Further experiments were conducted with addition of a second detergent, Zwittergent 3-14 which increased enzyme recovery to 45%. Lastly, reducing the concentrations of buffer and divalent cations Mn2+, Mg2+ and introducing glycerol (20%, v/v) allowed 80% of proteins to be solubilized together with 68% of the ceramide glucosyltransferase activity.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn(2+) for maximal activity. Optimum temperature was 42 degrees C. Apparent K(m) values for BH4 and UDP-glucose were determined as 4.3 microM and 188 microM, respectively, and V(max) values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.     

An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP-glucose:tetrahydrobiopterin glucosyltransferase

The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular disorders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP-glucose α-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP-glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin-glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4.     

Organization of mitochondrial UDP-glucose: Dolichylmonophosphate glucosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.     

Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves.

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12-18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.     

Carminic acid, a non-competitive inhibitor of kidney UDP-glucose:galactosylhydroxylysine-collagen glucosyltransferase

1. UDP-glucose:galactosylhydroxylsine-collagen glucosyltransferase was purified 12-fold from rat kidney. 2. An assay using calf-skin gelatin as substrate showed time- and enzyme-dependent incorporation; KmS for UDP-glucose and gelatin were 16-7 microM and 4.5 mg/ml, respectively. 3. Column chromatography of the alkaline hydrolysate of reaction product on Dowex 50W-4X(H+) showed that 84% of the radioactivity was in the glycosylgalactosylhydroxylsine peak. 4. Carminic acid inhibited collagen glycosyltransferase; a dose-dependent study showed a two-stage inhibition and kinetic analysis by double-reciprocal plots at varying UDP-glucose concentrations revealed a non-competitive mode of inhibition.     

Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase

Secretory and membrane N-linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP–glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP–glucose to N-linked Man9-GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9-GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino-acid residues close to N-linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli/

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067–1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.