Radioactive Sugars Research Articles

Glycosyltransferase Activity Assay Using Colorimetric Methods.

The glycosyltransferases (GTs) are an important subclass of enzymes that catalyze the biosynthesis of glycosidic bonds in oligosaccharides, polysaccharides and glycoconjugates by transferring a sugar residue from a donor substrate to an acceptor substrate. The membrane-associated GTs play a vital role in the biosynthesis of bacterial cell-wall polysaccharides. Characterization and quantification of GT activities is important for studies of biosynthesis of polysaccharides, drug target development, and production of bacterial products. In this chapter, colorimetric assays for the measurement of GT activities will be presented. Assays for GTs acting on monosaccharide-derivatives are based on the cleavage of unreacted glycosyl-p-nitrophenol acceptors followed by detection of p-nitrophenolate. GT reactions coupled with phosphatases and detection of inorganic phosphate are suitable for most GTs. These assays permit convenient quantification of GT activities and kinetics without the use of radioactive sugars.

The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5

The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-d-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars.

Teaching dolichol-linked oligosaccharides more tricks with alternatives to metabolic radiolabeling

The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol), transfer of G(3)M(9)Gn(2) to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [(3)H]-labeled or [(14)C]-labeled d-mannose, d-galactose, or d-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed.

Biosynthesis and cell-wall deposition of a pectin-xyloglucan complex in pea.

Golgi-enriched enzyme preparations prepared from etiolated pea epicotyls incorporated [U-(14)C]galactose from UDP-[U-(14)C]galactose into the 1,4-beta-galactan sidechains of a pectin-xyloglucan complex. This complex could bind to paper and was degraded both by pectin-degrading enzymes and by a xyloglucan-specific endoglucanase. Gel permeation chromatography was used to assess the molecular size of the complex and of enzymically-degraded, galactan-containing fragments of it. Etiolated pea stems were labelled with [U-(14)C]sucrose for 1 h, and the newly-synthesised cell wall polysaccharides were extracted with EDTA or NaOH and fractionated by ion-exchange chromatography. The NaOH-extracted, acidic radioactive polysaccharides obtained in this way were also degraded both by pectin-degrading enzymes and by xyloglucan-specific endoglucanase. Analysis of the radioactive sugar composition indicated that neutral sugars characteristic of both pectin and xyloglucan were present. Analysis of the total non-radioactive, neutral sugar composition of the NaOH-extracted, acidic cell-wall polysaccharides indicated that pectin-xyloglucan complexes were a general feature of the cell wall in this tissue.

Synthesis in vitro of novel nucleic-acid-like polymers of Ehrlich ascites tumor cells.

1 When cytoplasmic particles isolated from Ehrlich ascites tumor cells were incubated with [14C]ribose, es and inorganic phosphate in the presence of MgCl2 and Tris/HCl (pH 7.0), radioactivity was incorporated into trichloroacetic-acid-insoluble materials. 2 Radioactive products, extracted and purified by DEAE-cellulose column chromatography, were hydrolyzed in i M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were purified and identified as ribose, glucose and rhamnose by gas chromatography and mass spectrometry. 3 When [14C]glucose was incubated in the same reaction system, radioactivity was also incorporated into trichloroacetic-acid-insoluble materials. Purified radioactive products were hydrolyzed in 1 M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were also shown to be ribose, glucose and a little rhamnose, as was the case with the products synthesized from [14C]ribose. 4 When [14C]deoxyribose was incubated in the same reaction system, little incorporation was observed. 5 The radioactive products were incubated with venom phosphodiesterase and the digests were analyzed by Dowex lX4 column chromatography. The results suggest that the radioactive products contained ribo-nuclcotides and ‘nucleotides’ containing glucose (glucose ‘nucleotides’). 6 When the radioactive products were incubated with both venom phosphodiesterase and alkaline phosphatase, ribonucleosides and glucose ‘nucleosides’ were detected. In Dowex 1X4 (borate form) chromatography, tibonuclcosides were eluted coincidentalhy with ribonucleoside markers (Cyd Ado Urd and Guo), whereas the elution profiles of glucose ‘nucleosides’ were totallydifferent fromfacto of ribonucleosides.

N-Glycosylation of proteins by homogenate preparations of developing chick embryos.

Homogenate preparations of chick embryos contain the enzymes and endogenous precursors required for protein N-glycosylation mediated by dolichyl phosphate. The rate and extent of protein N-glycosylation was relatively high. Sensitivity of the first step in the dolichyl phosphate cycle to tunicamycin was confirmed. That tunicamycin had no indirect effect on the incorporation of glucose into lipid-linked intermediates or into protein but did reduce mannose incorporation was interpreted as supporting the view that the final glucosylation of the oligosaccharide is rate-limiting. Endogenous nucleotide diphosphate sugars and sugar acceptors, both lipid and protein linked, were present in the membrane preparation. Studies using dodecylsulphate-polyacrylamide gel electrophoresis on the radioactive glycoproteins formed during the incubations with nucleoside diphosphate [14C]sugars indicated that approximately one third of the 43 proteins detected carried radioactivity. Two thirds of the glycoproteins formed carried radioactive oligosaccharide chains that were either fully glucosylated or had been subsequently trimmed and received peripheral residues of N-acetylglucosamine. Most of the glycoproteins also carried radioactive oligosaccharide chains at varying stages of glycosylation preceding glucosylation and some carried only these ‘immature7rsquo; oligosaccharides. The transfer of mannose to protein and lipids was most active at 7 or 9 days of development being less active in both younger and older embryos whereas the pattern of change of activities of transfer of glucose and N-acetylglucosamine was more complicated. Most of the radioactive glycoproteins prominent at day 9 of development were also prominent in younger or older embryos. However, the incorporation of radioactive sugars into some glycoproteins, relative to that incorporated generally was very dependent upon the age of the embryos used.

Characteristics of the carbohydrate moiety and the intermolecular associations of the high-molecular-weight forms of alpha 2u-globulin.

We have investigated some of the biochemical characteristics of the glycosylated forms of α2u-globulin, the major protein of adult male rat urine. Labeled α2u-globulin that was secreted into the culture medium of primary monolayer cultures of hepatocytes was immunoprecipitated from the medium with antibody specific for urinary α2u-globulin and the complex used for further study. α2u-Globulin existed in the medium as a high-molecular-weight aggregate that could be dissociated with reducing agents and detergents into two glycosylated molecules of Mr 75000 and 50000 and one nonglycosylated molecule of Mr 20000. The glycosylated forms of α2u-globulin were shown by kinetic studies not to be precursors of the molecule of Mr 20000. When the glycoproteins were synthesized in the presence of radioactive sugar and amino acid precursors, the ratio of sugar to amino acid suggested that the highest-molecular-weight glycoprotein had 1.5–2 times the carbohydrate content of the faster-migrating glycoprotein. The oligosaccharide moiety, isolated from an exhaustive pronase digest of the combined glycoprotein molecules, was of Mr 5000 as determined by gel nitration methods. Sequential glycosidase digestion with neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase produced a core oligosaccharide of Mr 1200. Anion-exchange chromatography demonstrated that the charge microheterogeneity of the α2u-globulin glycopeptides could be abolished by treatment with neuraminidase. Our results indicate that the oligosaccharide moiety of α2u-globulin is similar to those oligosaccharides found on other serum proteins, and suggest that formation of the species of α2u-globulin of Mr 75000 and 50000 requires either the attachment of many oligosaccharide chains to a single polypeptide of Mr 20000 or the covalent linkage of poly-peptides containing one or more oligosaccharide chains to form dimers and trimers.

Fluorophore-assisted carbohydrate electrophoresis: a sensitive and accurate method for the direct analysis of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues

Lipid-linked oligosaccharides (LLOs) such as Glc 3Man 9GlcNAc 2-P-P-dolichol are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I Congenital Disorders of Glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [ 3H]mannose or [ 14C]glucosamine. In this article, a procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO analysis based on fluorophore-assisted carbohydrate electrophoresis (FACE). First, LLOs are extracted and partially purified. Next, oligosaccharides released from LLOs are labeled with negatively charged fluorophores: 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) or 7-amino-1,3-naphthalenedisulfonic acid (ANDS). A specialized form of polyacrylamide gel electrophoresis is then used to resolve and measure ANTS or ANDS labeled oligosaccharides. Finally, the resolved oligosaccharides are detected and quantified by fluorescence imagers using CCD cameras.

A novel enzymatic reaction for converting DNA to CO-DNA

We have found previously that DNA from both the chick cerebrum and cardiac muscle has a modified structure. We named this novel DNA, CO-DNA. CO-DNA is a form of DNA in which a carbonyl group is attached to C-1 of the 2-deoxyribose and to the nitrogenous base. Therefore, 3-deoxyglucosone is the sugar constituent for CO-DNA. We found previously that the modification of the sugar moiety in DNA occurs around embryonic day 12 in the chick embryo. In this study, we isolated enzymes for the conversion of DNA to CO-DNA from chick cerebra. In our reaction system, uniformly labeled 14C-glucose was used as substrate. During incubation, the radioactivity was incorporated into DNA. From the analysis of 14C-labeled deoxynucleoside, the radioactive sugar was confirmed to be 3-deoxyglucosone. We propose a series of reactions involved in the conversion of DNA to CO-DNA: (1) DNA–enzyme complex is formed during preincubation, (2) 14C-glucose is transformed to 14C-3-deoxyhexonic acid, (3) 14C-3-deoxyhexonic acid is subsequently transformed into the sugar-phosphate, which is a mixture of phosphorylated 14C-3-deoxyhexonic acid and phosphorylated 14C-3-deoxyglucosone, (4) 2-deoxyribose in DNA is replaced with 14C-3-deoxyglucosone through its intermediate phosphorylated form, and (5) DNA is finally converted to CO-DNA.

Glycosyltransferase assays utilizing N-acetyllactosamine acceptor immobilized on a cellulose membrane

Solid-phase assays for measuring the activity of four different glycosyltransferase enzymes that utilize N-acetyllactosamine as an acceptor are reported. These enzymes are α1,3-galactosyltransferase (E.C. 2.4.1.151), α1,3-fucosyltransferase (E.C. 2.4.1.65), α2,6-( N)-sialyltransferase (E.C. 2.4.99.1), and α2,3-( N)-sialyltransferase (E.C. 2.4.99.5). The acceptor is immobilized on a cellulose membrane in two different ways, through either an amine-cleavable linker or a photolinker. Incubation with a glycosyltransferase and nucleotide donor sugar resulted in the transfer of a monosaccharide from the donor to immobilized N-acetyllactosamine. For galactosyltransferase, transfer was confirmed by mass spectrometry of the products cleaved from the membrane surface after amine treatment or irradiation. When radioactive donors were utilized, the transfer of radioactive sugars could be monitored by autoradiography. Alternatively the transfer of radioactive sugar onto the membranes could be measured by scintillation counting of the products after cleavage from the membrane. Cytidine 5 ′-monophosphate–sialic acid carrying a fluorescent tag in the saccharide was also successfully utilized in this assay system. Fluorescent product on the membrane surface was detected by imaging. Glycosyltransferase assays with these versatile membranes have the potential to be adapted for high-throughput screening.

Functional asymmetry of the human Na+/glucose transporter (hSGLT1) in bacterial membrane vesicles.

The functional characteristics of the forward and reverse transport modes of the human Na(+)/glucose transporter (hSGLT1) were investigated using plasma membrane vesicles of E. coli expressing the recombinant transporter. Correctly and inverse-oriented vesicles were employed to measure the initial rates of methyl-alpha-D-glucose uptake, under zero-trans conditions, as a function of Na(+), sugar, and phlorizin concentrations and membrane potential. This approach enabled the analysis of the two faces of hSGLT1 in parallel, revealing the reversibility of Na(+)/sugar cotransport. While the key characteristics of secondary active sugar transport were maintained in both modes, namely, Na(+) and voltage dependence, the kinetic properties of the two sides indicated a functional asymmetry of the transporter. That is, the apparent affinity for sugar and driver cation Na(+) exhibited a difference of more than 1 order of magnitude between the two modes. Furthermore, the selectivity pattern of ligands and the interaction of the transporter with the competitive inhibitor phlorizin were different. Whereas the high-affinity substrates, D-glucose and D-galactose, inhibited uptake of radioactive sugar tracer at their physiological concentrations (10 mM) in the forward reaction, they were poor inhibitors even at high concentrations in the reverse transport mode. Taken together, these results confirm the successful employment of E. coli to express and characterize a human membrane protein (hSGLT1), elucidating the functional asymmetry of this cotransporter.

Increased Biosynthesis of Glycosphingolipids in Congenital Disorder of Glycosylation Ia (CDG-Ia) Fibroblasts

Congenital disorder of glycosylation Ia (CDG-Ia) is an autosomal recessive disease, characterized by the impaired biosynthesis of the N-linked oligosaccharide chains of proteins due to a deficiency of phosphomannomutase (PMM), the enzyme converting mannose-6-phosphate into mannose-1-phosphate. We investigated the consequences of the altered N-linked glycoprotein (GP) biosynthesis on the quantity and quality of glycosphingolipids (GSLs) in fibroblasts of CDG-Ia patients. First, we found that CDG-Ia fibroblasts contain an increased amount of total GSLs when compared with normal fibroblasts. Further, we assessed by metabolic labeling of CDG-Ia fibroblasts with radioactive sugar precursors, including galactose and N-acetylmannosamine, that a diminished biosynthesis of cellular GPs is antagonized by an increased biosynthesis of GSLs. An increased GSL biosynthesis was also observed by means of radiolabeled lipid precursors including sphingosine and lactosylceramide. Notably, also the degradation of GLSs is slowed down in CDG-Ia fibroblasts. Finally, when we labeled normal human fibroblasts and CHO cells with radioactive galactose in the presence and absence of deoxymannojirimycin (dMM), an inhibitor of N-glycan processing, we found that this cellular model mimics what occurs in CDG-Ia fibroblasts. Since an inverse relationship between GP expression and GSL content does exist, we assume that increased glycosphingolipid biosynthesis is secondary to protein hypoglycosylation. Altogether, our data suggest that the cell metabolic machinery may be able to partially re-equilibrate protein hypoglycosylation with increased biosynthesis of glycosphingolipids, possibly to preserve the overall physico-chemical equilibrium of the outer layer of the plasma membrane.

Coupling of the Dolichol-P-P-Oligosaccharide Pathway to Translation by Perturbation-sensitive Regulation of the Initiating Enzyme, GlcNAc-1-P Transferase

In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.

Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis.

Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.

Identification and Molecular Cloning of a Heparosan Synthase fromPasteurella multocida Type D

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.

Biosynthesis of d-Arabinose in Mycobacterium smegmatis: Specific Labeling from d-Glucose

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-14C]glucose, [3,4-14C]glucose, and [6-14C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-14C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-13C]glucose, [2-13C]glucose, and [6-13C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative 13C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [14C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [3H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.

Metabolic Labeling of Glycoproteins with Radioactive Sugars

This unit describes methods for preparation of glycoproteins metabolically labeled with radioactive sugars, sulfate, and phosphate. Methods for liberation of both N- and O-linked glycans are also described. These protocols can be used to generate materials for characterization of glycoprotein glycans from cultured cells.

Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929–939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH2 and -CO2H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH2 (GelKNH2) and -CO2H (GelKCOOH) halves were overexpressed in Escherichia coli. GelK and GelKNH2 were present in both the soluble and membrane fractions of E. coli, whereas GelKCOOH was only present in the soluble fraction. GelK catalysed the transfer of [14C]glucuronic acid from UDP-[14C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by β-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activity. Recombinant GelKNH2 and GelKCOOH did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.