Cell-surface glycosyltransferases in gastrulating chick embryos. I. Temporally and spatially specific patterns of four endogenous glycosyltransferase activities.

Seven classes of endogenous cell-surface glycosyltransferase activities have been investigated in gastrulating chick embryos. Galactosyl-, N-acetylglucosaminyl-, fucosyl-, and sialyl-transferases show temporally and spatially specific autoradiographic patterns that are characteristic for each transferase class. Invaginating primitive streak cells, primordial germ cells, and cranial neural crest cells demonstrate some of the most intense extracellular glycosyltransferase activity. Glucosyl-, N-acetylgalactosaminyl-, and glucuronyltransferases are relatively inactive under these assay conditions. A model is proposed that suggests that embryonic cells migrate over carbohydrate substrates via surface transferase binding to exposed oligosac-charide side chains. One prediction of this model is that sugar nucleotides may be teratogenic. Preliminary experiments support such a prediction. Embryos incubated with UDPgal and UDPglcNAc develop abnormally in ways that are consistent with the locations of some transferase classes. The free sugars, galactose, and N-acetylglucosamine, as well as one of the inactive sugar nucleotides, are relatively ineffective in disturbing normal morphogenesis.     

Nucleotide sugars and glycosyltransferases for synthesis of cell wall matrix polysaccharides

The current interest in cell wall biosynthesis is expanding because of the increasing evidence that the properties of the cell wall mediate cellular interactions during growth, development and differentiation. Much effort has been put forward to the identification of glycosyltransferases because of their obvious importance in polysaccharide synthesis. Enzymes involved in nucleotide sugar production and transport are also important because of the potential to manipulate the composition of cell walls through substrate level control. Molecular genetics have begun to uncover genes for important enzymes in polysaccharide biosynthesis including glycosyltransferases and enzymes of nucleotide sugar metabolism; but at this time, much is inferred from comparisons to bacteria, yeast and animal cells. This review examines the production and transport of nucleotide sugars, the protein structure of glycosyltransferases, and implications for the cellular mechanisms of cell wall biosynthesis.     

Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases

An effective procedure for the synthesis of 2-deoxy-2-fluoro-sugar nucleotides via Select fluor-mediated electrophilic fluorination of glycals with concurrent nucleophilic addition or chemo-enzymatic transformation has been developed, and the fluorinated sugar nucleotides have been used as probes for glycosyltransferases, including fucosyltransferase III, V, VI, and VII, and sialyl transferases. In general, these fluorinated sugar nucleotides act as competitive inhibitors versus sugar nucleotide substrates and form a tight complex with the glycosyltransferase.     

Cell surface glycosyltransferases—do they exist?

The presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: (1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and (2) loss of cell integrity if trypsinized or EDTA-treated cells were used in suspension asays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl-and sialyltransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.     

Method for electroporation for the early chick embryo

In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.     

Developmental changes in collagen glycosyltransferase activities in chick embryos

The developmental pattern of collagen galactosyltransferase and collagen glucosyltransferase activities was determined in chick embryos between the 4th and 21st day of growth. Both enzyme activities increased up to the 16th day and decreased thereafter in whole chick embryos and in most tissues studied. The highest collagen glycosyltransferase activities were found in the leg tendons of the 16-day-old embryos, and the activities found in cartilage were higher than those noted in either skin or skull, indicating that the the activities of the collagen glycosyltransferases may play a part in the regulation of the carbohydrate content of the collagen synthesized by a given tissue. The changes observed in the collagen glycosyltransferase activities agree with previous data on the development of prolyl and lysyl hydroxylase activities and also with findings on collagen turnover in the developing chick embryo.     

Features and applications of bacterial glycosyltransferases: current state and prospects

The bioactivity of many natural products including valuable antibiotics and anticancer therapeutics depends on their sugar moieties. Changes in the structures of these sugars can deeply influence the biological activity, specificity and pharmacological properties of the parent compounds. The chemical synthesis of such sugar ligands is exceedingly difficult to carry out and therefore impractical to establish on a large scale. Therefore, glycosyltransferases are essential tools for chemoenzymatic and in vivo approaches for the development of complex glycosylated natural products. In the last 10 years, several examples of successful alteration and diversification of natural product glycosylation patterns via metabolic pathway engineering and enzymatic glycodiversification have been described. Due to the relaxed substrate specificity of many sugar biosynthetic enzymes and glycosyltransferases involved in natural product biosynthesis, it is possible to obtain novel glycosylated compounds using different methods. In this review, we would like to provide an overview of recent advances in diversification of the glycosylated natural products and glycosyltransferase engineering.     

Thin-layer chromatographic method for the determination of glycosyltransferase activity

To survey glycosyltransferase activities and specificities we have developed a TLC method to separate various nucleotide sugars from both high- and low-molecular-weight sugar acceptors. Here, we report details of the procedure and its application for galactosyltransferase and fucosyltransferase detected in mouse spermatogenic cells. The assay method involves sample separation using polyethyleneimine cellulose plastic-backed thin-layer plates, developed in sodium phosphate buffer for 30 min. Nucleotide sugars, including UDP-Gal, GDP-Fuc, CMP-NeuNAc, and GDP-Man, remain at the origin, while both high- and low-molecular-weight sugar acceptors migrate within 2 cm of the solvent front. Assays for galactosyltransferase and fucosyltransferase are linear with time and yield results comparable to other methods such as gel permeation chromatography and micropartitioning filtration. The TLC protocol should be useful for determinations of many different glycosyltransferases.     

Nucleotide sugar transporters: biological and functional aspects

The Golgi apparatus serves as the major site of glycosylation reactions. Nucleotide sugars which are substrates of the Golgi localized glycosyltransferases are synthesized in the cytoplasm (cell nucleus in case of CMP-sialic acid) and must be transported into the compartment lumen. This transport function is carried out by nucleotide sugar transporters. The first genes were cloned in the year 1996 and revealed a family of structurally conserved multi-transmembrane-spanning proteins. Due to the high structural and functional conservation, the identification of many putative nucleotide sugar transporter sequences has become possible in the existing gene data bases and accelerates the increase in knowledge on structure-function-relationships. Recent developments in the nucleotide sugar transporter field are discussed in this article.     

A bacterial glycosyltransferase gene toolbox: Generation and applications

The bioactivity of many natural products produced by microorganisms can be attributed to their sugar substituents. These substituents are transferred as nucleotide-activated sugars to an aglycon by glycosyltransferases. Engineering these enzymes can broaden their substrate specificity and can therefore have an impact on the bioactivity of the secondary metabolites.In this review we present the generation of a glycosyltransferase gene toolbox which contains more than 70 bacterial glycosyltransferases to date. Investigations of the function, specificity and structure of these glycosyltransferases help to understand the great potential of these enzymes for natural product biosynthesis.     

微生物合成黄酮糖苷类天然产物研究进展

黄酮糖苷类天然产物是植物中黄酮类化合物的主要存在形式,通过糖基化修饰,可以改变其水溶性、稳定性等,赋予其新的生物活性和功能。黄酮类化合物的糖基化修饰通常由植物源或微生物源的糖基转移酶催化,根据糖基的位置、类型和数量的不同,可形成多种类型的黄酮糖苷类产物。随着合成生物学和代谢工程的快速发展,在微生物中合成植物源黄酮糖苷类天然产物取得了重要进展。综述了糖基转移酶的聚类分析及糖基供体的途径改造,并对代谢工程优化黄酮糖苷类天然产物的微生物合成进行了分析讨论,并对其发展前景进行了展望。     

Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars: I. Determination of the degradation rate during the glycosyltransferase assays

In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phophatases; 3, chemical decomposition of UDP-D-[¹⁴C]Gal; UDP-D-[¹⁴C]Glc and UDP-D-[¹⁴C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide.All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined.Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn²⁺, Mg²⁺). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate.A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP-D-Gal. The results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.     

Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)

Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.     

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.     

The Localization and Potential Function of Glycosyltransferases in Chick Embryos

Intact, developing chick embryos (stages 9+ to 14) incorporatesugars from sugar nucleotides in patterns which vary accordingto the sugar nucleotide used, the area of the embryo, and theage of the embryo. Radioautographs of the notochord-somite-lateralplate mesoderm vicinity, the optic vesicle-skin ectoderm junction,and the area vasculosa are presented. It is possible that thesepatterns represent localized cell surface glycosyltransferase-glycosylacceptor complexes. However, other possibilities have not beenruled out. The potential role of enzyme-substrate complexesin intercellular interactions is discussed.     

常见致病菌糖基转移酶的结构与功能

糖基转移酶(glycosyltransferases,GTs)将糖基从活化的供体转移到糖、脂、蛋白质和核酸等受体,其参与的蛋白质糖基化是最重要的翻译后修饰(post-translational modifications,PTMs)之一。近年来越来越多的研究证明,糖基转移酶与致病菌毒力密切相关,在致病菌的黏附、免疫逃逸和定殖等生物学过程中发挥关键作用。目前,已鉴定的糖基转移酶根据其蛋白质三维结构特征分为3种类型GT-A、GT-B和GT-C,其中常见的是GT-A和GT-B型。在致病菌中发挥黏附功能的糖基转移酶,在结构上属于GT-B或GT-C型,对致病菌表面蛋白质(黏附蛋白、自转运蛋白等)进行糖基化修饰,在致病菌黏附、生物被膜的形成和毒力机制发挥具有重要作用。糖基转移酶不仅参与致病菌黏附这一感染初始过程,其中属于GT-A型的一类致病菌糖基转移酶会进入宿主细胞,通过糖基化宿主蛋白质影响宿主信号传导、蛋白翻译和免疫应答等生物学功能。本文就常见致病菌糖基转移酶的结构及其糖基化在致病机制中的作用进行综述,着重介绍了特异性糖基化高分子量(high-molecular-weight,HMW)黏附蛋白的糖基转移酶、针对富丝氨酸重复蛋白(serine-rich repeat proteins,SRRP)糖基化修饰的糖基转移酶、细菌自转运蛋白庚糖基转移酶(bacterial autotransporter heptosyltransferase,BAHT)家族、N-糖基化蛋白质系统和进入宿主细胞发挥毒力作用的大型梭菌细胞毒素、军团菌(Legionella)葡萄糖基转移酶以及肠杆菌科的效应子NleB。为揭示致病菌中糖基转移酶致病机制的系统性研究提供参考,为未来致病菌的诊断、药物设计研发以及疫苗开发等提供科学依据和思路。     

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.     

Biosynthesis of chondroitin sulfate proteoglycan. Subcellular distribution of glycosyltransferases in embryonic chick brain.

The subcellular distributions of five glycoslytransferases involved in the biosynthesis of the chondroitin sulfate proteoglycan and of a sixth glycosyltransferase, presumably involved in glycoprotein biosynthesis, were examined in 13-day chick enbryo brain. Fractionation studies performed by the procedure of Gray and Whittaker (Gray, E.G., and Whittaker, V.P. (1962) J. Anat. (London) 96, 79-88) revealed that three of the six enzymes were directly associated with the membrane fraction of synaptosome-enriched preparations; varying amounts of the remaining glycosyltransferases were distributed between the 100,000 times g supernatant and the synaptosome-enriched fraction after differential and density gradient centrifugation of crude chick brain homogenates. The time of appearance of three of the glycosyltransferases was examined in chick embryo brain tissue at several stages of development. The brain content of each glycosyltransferase increased rapidly between day 7 and hatching at day 21. A sharp decline in each of the glycosyltransferase activities occurred at hatching.     

The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles.

In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.     

The relationship between the branch-forming glycosyltransferases and cell surface sugar chain structures

Many recombinant proteins developed or under development for clinical use are glycoproteins, and trials aimed at improving their bioactivity or pharmacokinetics in vivo by altering specific glycan structures are ongoing. For pharmaceuticals of glycoproteins, it is important to characterize and, if possible, control the glycosylation profile. However, the mechanism responsible for the regulation of sugar chain structures found on naturally occurring glycoproteins is still unclear. To clarify the relationship between glycosyltransferases and sugar chain branch structure, we estimated six glycosyltransferases' activities (N-acetylglucosaminyltransferase (GlcNAcTase)-I, -II, -III, -IV, -V, and beta-1,4-galactosyltransferase (GalT)) which control the branch formation on asparagine (Asn)-linked sugar chains in 18 human cancer cell lines derived from several tissues. To visualize the balance of glycosyltransferase activity associated with each cell line, we expressed the relative glycosyltransferase activity in comparison to the average activity among the cell lines. These cell lines were classified into five groups according to their relative glycosyltransferase balance and were termed GlcNAcTase-I/-II, GlcNAcTase-III, GlcNAcTase-IV, GlcNAcTase-V, and GalT. We also characterized the structures of Asn-linked sugar chains on the cell surface of representative cell lines of each group. The branching structure of cell surface sugar chains roughly corresponded to the glycosyltransferase balance. This finding suggests that, for the sugar chain structure remodeling of glycoproteins, attention should be focused on the glycosyltransferase balance of host cells before introducing exogenous glycosyltransferases or down-regulating the activity of intrinsic glycosyltransferases.