A fluorescence-based glycosyltransferase assay for high-throughput screening

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.     

Insights into O-linked N-acetylglucosamine ([0-9]O-GlcNAc) processing and dynamics through kinetic analysis of O-GlcNAc transferase and O-GlcNAcase activity on protein substrates

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.     

Glycoprotein metabolism in rat colonic epithelial cell populations with different proliferative activities

Abstract. Epithelial cells with different proliferative activities were isolated from rat proximal and distal colon. The distribution of fucose, hexose, hexosamine, and sialic acid as well as the activities of three glycosyltransferases, eight glycosidases, and a nucleotide sugar pyrophosphatase, enzymes involved in glycoprotein metabolism, were then examined in these cells. The results of the present study demonstrate that: (1) all proximal cell populations appear to possess a higher content of hexose, fucose, and sialic acid than their distal counterparts; (2) in general, the proximal colonic populations have higher glycosyltransferase but similar glycosidase activities than their distal counterparts; (3) proliferative cells in both colonic regions have greater glycosyltransferase and glycosidase activities than non-proliferative cells, although their carbohydrate content is similar.
These findings suggest that alterations in glycoprotein metabolism exist during differentiation along the length of the rat colon. Furthermore, these data indicate that certain enzymes involved in glycoprotein metabolism may serve as markers for cellular differentiation in this organ.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Abstract Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

Structure/function analysis of Pasteurella multocida heparosan synthases: toward defining enzyme specificity and engineering novel catalysts

The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (～65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyl transferase

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.     

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.     

Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs

Many bacterial pathogens express lipooligosaccharides that mimic human cell surface glycoconjugates, enabling them to attach to host receptors and to evade the immune response. In Neisseria meningitidis, the galactosyltransferase LgtC catalyzes a key step in the biosynthesis of lipooligosaccharide structure by transferring alpha-d-galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the donor sugar glycosidic bond; LgtC is thus a retaining glycosyltranferase. We report the 2 A crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose (a donor sugar analog) in the presence and absence of the acceptor sugar analog 4'-deoxylactose. The structures, together with results from site-directed mutagenesis and kinetic analysis, give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.     

O-linked N-acetylglucosaminyltransferase inhibition prevents G2/M transition in Xenopus laevis oocytes

Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G(2)-like state. Progesterone triggers meiotic resumption also called the G(2)/M transition. This event is characterized by germinal vesicle breakdown (GVBD) and by a burst in phosphorylation level that reflects activation of M-phase-promoting factor (MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G(2)/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes (SOD and catalase) did not rescue GVBD, indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened by the use of a benzoxazolinone derivative (XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine:fructose-6-phosphate amidotransferase inhibitors, azaserine and 6-diazo-5-oxonorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G(2)/M transition and strongly suggests that its function is required for cell cycle regulation.     

Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate

A highly sensitive fluorogenic hexosaminidase substrate, fluorescein di(N-acetyl-beta-D-glucosaminide) (FDGlcNAc), was prepared essentially as described previously [Chem. Pharm. Bull. 1993, 41, 314] with some modifications. The fluorescent analog is a substrate for a number of hexosaminidases but here we have focused on the cytoplasmic O-GlcNAcase isoforms. Kinetic analysis using purified O-GlcNAcase and its splice variant (v-O-GlcNAcase) expressed in Escherichia coli suggests that FDGlcNAc is a much more efficient substrate (Km = 84.9 microM) than the conventional substrate, para-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (pNP-beta-GlcNAc, Km = 1.1 mM) and a previously developed fluorogenic substrate, 4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside [MUGlcNAc, Km = 0.43 mM; J. Biol. Chem. 2005, 280, 25313] for O-GlcNAcase. The variant O-GlcNAcase, a protein lacking the C-terminal third of the full-length O-GlcNAcase, exhibited a Km of 2.1 mM with respect to FDGlcNAc. This shorter isoform was not previously thought to exhibit O-GlcNAcase activity based on in vitro studies with pNP-beta-GlcNAc. However, both O-GlcNAcase isoforms reduced O-GlcNAc protein levels extracted from HeLa and HT-29 cells in vitro, indicating that the splice variant is a bona fide O-GlcNAcase. Fluorescein di-N-acetyl-beta-D-galactosaminide (FDGalNAc) is not cleaved by these enzymes, consistent with previous findings that the O-GlcNAcase has substrate specificity toward O-GlcNAc but not O-GalNAc. The enzymatic activity of the shorter isoform of O-GlcNAcase was first detected by using highly sensitive fluorogenic FDGlcNAc substrate. The finding that O-GlcNAcase exists as two distinct isoforms has a number of important implications for the role of O-GlcNAcase in hexosamine signaling.     

Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F

Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.     

Synthesis and rapid purification of UDP-[6-3H]galactose

UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.     

Purification and identification of dTDP-oleandrose, the precursor of the oleandrose units of the avermectins

The nucleotide sugar precursor of the oleandrose units of the avermectins has been purified from a mutant of Streptomyces avermitilis, which does not synthesize any avermectins but which converts avermectin aglycones to their respective disaccharides. This precursor has been identified as dTDP-oleandrose. The purification was achieved by anion exchange and reverse phase high performance liquid chromatography. The purified nucleotide sugar had an absorption spectra characteristic of thymidine, released dTMP when treated with phosphodiesterase, and possessed an NMR spectrum in which three resonances characteristic of oleandrose were seen in addition to the thymidine signals. The enzyme, avermectin aglycone dTDP-oleandrose glycosyltransferase, which catalyzes the stepwise addition of oleandrose to the avermectin aglycones, has been demonstrated in cell-free extracts and (NH4)2SO4 fractions of cell-free extracts of S. avermitilis. The enzyme is specific for dTDP-oleandrose as the glycosyl donor but utilizes all avermectin aglycones as glycosyl acceptors. The stoichiometry between dTDP-oleandrose consumed in the reaction and oleandrose units transferred to the avermectin mono- and disaccharide was found to be 1:1.