Structural and functional features of glycosyltransferases

Most of the glycosylation reactions that generate the great diversity of oligosaccharide structures of eukaryotic cells occur in the Golgi apparatus. This review deals with the most recent data that provide insight into the functional organization of Golgi-resident glycosyltransferases. We also focus on the recent successes in X-ray crystal structure determination of glycosyltransferases. These new structures begin to shed light on the molecular bases accounting for donor and acceptor substrate specificities as well as catalysis.     

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。     

Structure/function relationship of homopolysaccharide producing glycansucrases and therapeutic potential of their synthesised glycans

The capability of lactic acid bacteria (LAB) to produce exopoly- and oligosaccharides was and is the subject of expanding research efforts. Due to their physicochemical properties and health-promoting potential, exopoly- and oligosaccharides from food-grade LAB can be used in the food and other industries and may have additional medical applications. In the last years, many LAB have been screened for their ability to produce exopoly- and oligosaccharides, and several glycosyltransferases involved in their biosynthesis have been characterised at biochemical and genetic levels. These research efforts aim to exploit the full potential of these organisms and to understand the structure/function relationship of glycosyltransferases. The latter knowledge is a prerequisite for the production of tailored exopoly- and oligosaccharides for the diverse applications. This review will survey the results of recent works on the structure/function relationship of homopolysaccharide producing glycosyltransferases and the therapeutic potential of their synthesised exopoly- and oligosaccharides.     

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.     

Glycosyltransferases involved in the biosynthesis of biologically active natural products that contain oligosaccharides

A unique characteristic of carbohydrates is their structural diversity which is greater than that of many other classes of biological compounds. Carbohydrate-containing natural products show many different biological activities and some of them have been developed as drugs for medical use. The biosynthesis of carbohydrate-containing natural products is catalysed by glycosyltransferases. In this review we will present information on the function of glycosyltransferases involved in the biosynthesis of oligosaccharide antibiotics focusing especially on urdamycins and landomycins, two angucycline antibiotics with interesting antitumor activities. We will also discuss the use of glycosyltransferases in combinatorial biosynthesis to generate new "hybrid" antibiotics.     

植物激素糖基化修饰研究进展

植物激素对植物的生长发育有重要的调节作用。由于激素的作用依赖于其浓度, 所以植物内源活性激素的水平必须受到严格控制, 而糖基化修饰被认为是调控激素活性水平的重要方式之一。随着植物激素糖基化修饰相关糖基转移酶基因不断被克隆与鉴定, 多种植物激素的糖基化修饰机制和功能作用逐渐被揭示。该文重点介绍了近年来植物生长素、细胞分裂素、脱落酸、油菜素内酯、水杨酸、茉莉酸等植物激素的糖基转移酶活性鉴定与功能研究进展。同时, 对植物激素糖基化修饰领域存在的问题和发展前景进行了讨论。     

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.     

Glycosyltransferases and their products: cryptococcal variations on fungal themes

Glycosyltransferases are specific enzymes that catalyse the transfer of monosaccharide moieties to biological substrates, including proteins, lipids and carbohydrates. These enzymes are present from prokaryotes to humans, and their glycoconjugate products are often vital for survival of the organism. Many glycosyltransferases found in fungal pathogens such as Cryptococcus neoformans do not exist in mammalian systems, making them attractive potential targets for selectively toxic agents. In this article, we present the features of this diverse class of enzymes, and review the fungal glycosyltransferases that are involved in synthesis of the cell wall, the cryptococcal capsule, glycoproteins and glycolipids. We specifically focus on enzymes that have been identified or studied in C. neoformans, and we consider future directions for research on glycosyltransferases in the context of this opportunistic pathogen.     

The yeast expression system for recombinant glycosyltransferases

Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include beta1,4galactosyltransferase, alpha2,6sialyltransferase, alpha2,3sialyltransferase, alpha1,3fucosyltransferase III and VI and alpha1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.     

Enzyme-catalyzed oligosaccharide synthesis.

Cell-surface carbohydrates and their conjugates are involved in many types of molecular recognition. This review describes recent developments in enzyme-catalyzed oligosaccharide synthesis, with particular focus on glycosyltransferase and glycosidase reactions. With the increasing availability of glycosyltransferases via recombinant DNA technology, glycosyltransferase-catalyzed glycosylation with in situ regeneration of sugar nucleotides appears to be the most effective method for large-scale stereocontrolled oligosaccharide synthesis.     

Advances in flavonoid glycosyltransferase research: integrating recent findings with long-term citrus studies

Flavonoid glycosides are required for a number of crucial roles in planta and have the potential for development in a variety of agricultural, medicinal, and biotechnological applications. A number of recent advancements have been made in characterizing glycosyltransferases, the enzymes that are responsible for the synthesis of these important molecules. In this review, glycosyltransferases are considered with regard to biochemical properties, expression patterns, levels of enzyme activity during development, and structure/function relationships. This is presented with historical context to highlight critical findings, particularly with regard to the innovative work that has come from research on citrus species. The plant glycosyltransferase crystal structures that have been solved over the past decade, either alone or in complex with sugar donor and/or acceptor molecules, are discussed. The application of results from these structures to inform current structure/function work as well as implications and goals for future crystallography and tertiary modeling studies are considered. A thorough understanding of the properties of glycosyltransferases will be a critical step in any future biotechnological application of these enzymes in areas such as crop improvement and custom design of enzymes to produce desired compounds for nutritional and/or medicinal usage.     

Conserved domains of glycosyltransferases.

Glycosyltransferases catalyze the synthesis of glycoconjugates by transferring a properly activated sugar residue to an appropriate acceptor molecule or aglycone for chain initiation and elongation. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme. To elucidate the structural requirements for substrate recognition and catalytic reactions of glycosyltransferases, we have searched the databases for homologous sequences, identified conserved amino acid residues, and proposed potential domain motifs for these enzymes. Depending on the configuration of the anomeric functional group of the glycosyl donor molecule and of the resulting glycoconjugate, all known glycosyltransferases can be divided into two major types: retaining glycosyltransferases, which transfer sugar residue with the retention of anomeric configuration, and inverting glycosyltransferases, which transfer sugar residue with the inversion of anomeric configuration. One conserved domain of the inverting glycosyltransferases identified in the database is responsible for the recognition of a pyrimidine nucleotide, which is either the UDP or the TDP portion of a donor sugar-nucleotide molecule. This domain is termed "Nucleotide Recognition Domain 1 beta," or NRD1 beta, since the type of nucleotide is the only common structure among the sugar donors and acceptors. NRD1 beta is present in 140 glycosyltransferases. The central portion of the NRD1 beta domain is very similar to the domain that is present in one family of retaining glycosyltransferases. This family is termed NRD1 alpha to designate the similarity and stereochemistry of sugar transfer, and it consists of 77 glycosyltransferases identified thus far. In the central portion there is a homologous region for these two families and this region probably has a catalytic function. A third conserved domain is found exclusively in membrane-bound glycosyltransferases and is termed NRD2; this domain is present in 98 glycosyltransferases. All three identified NRDs are present in archaebacterial, eubacterial, viral, and eukaryotic glycosyltransferases. The present article presents the alignment of conserved NRD domains and also presents a brief overview of the analyzed glycosyltransferases which comprise about 65% of all known sugar-nucleotide dependent (Leloir-type) and putative glycosyltransferases in different databases. A potential mechanism for the catalytic reaction is also proposed. This proposed mechanism should facilitate the design of experiments to elucidate the regulatory mechanisms of glycosylation reactions. Amino acid sequence information within the conserved domain may be utilized to design degenerate primers for identifying DNA encoding new glycosyltransferases.     

Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions

The glycoproteins of tumour cells are often abnormal, both in structure and in quantity. In particular, the mucin-type O-glycans have several cancer-associated structures, including the T and Tn antigens, and certain Lewis antigens. These structural changes can alter the function of the cell, and its antigenic and adhesive properties, as well as its potential to invade and metastasize. Cancer-associated mucin antigens can be exploited in diagnosis and prognosis, and in the development of cancer vaccines. The activities and Golgi localization of glycosyltransferases are the basis for the glycodynamics of cancer cells, and determine the ranges and amounts of specific O-glycans produced. This review focuses on the glycosyltransferases of colon and breast cancer cells that determine the pathways of mucin-type O-glycosylation, and the proposed functional and pathological consequences of altered O-glycans.     

Masking and reduction of the Galactose-alpha1,3-Galactose (alpha-Gal) epitope, the major xenoantigen in swine, by the glycosyltransferase gene transfection.

The alpha-Gal epitope (Gal-alpha1-3Gal-beta1-4-GlcNAc-R), which is biosynthesized by the UDP-Gal:alpha1-3-galactosyltransferase (alpha1, 3GT), is highly associated with hyperacute rejection in swine to human xenotransplantation. A variety of strategies have been pursued to reduce or eliminate this epitope from swine tissues. Since swine ES cells are not available at present, the targeted knock out of the alpha1,3GT is restricted. Other strategies, such as enzyme competition of the alpha1,3GT with other glycosyltransferases and/or control of sugar processing by the glycosyltransferases, provide a new insight into the downregulation of the alpha-Gal epitope. This review will focus on this type of strategy, which involves a gene transfection of variety of glycosyltransferases as competitors against alpha1,3GT.     

Enzymatic tools for engineering natural product glycosylation

Glycosylated natural products have served as reliable platforms for the development of many existing front-line drugs. In an effort to explore the contribution of the sugar constituents of these compounds, research groups have focused upon the development of chemical and enzymatic tools to diversify natural product glycosylation. Among the complementary routes available, in vivo pathway engineering, also referred to as 'combinatorial biosynthesis', is an emerging method that relies upon the co-expression of sugar biosynthetic gene cassettes and glycosyltransferases in a host organism to generate novel glycosylated natural products. An overview of recent progress in combinatorial biosynthesis is highlighted in this review, emphasizing the elucidation of nucleotide-sugar biosynthetic pathways and recent developments on glycosyltransferases.     

Regulation of expression of carbohydrate blood group antigens

The carbohydrate antigens associated with the human ABO and Lewis blood group systems are excellent models for the study of the genetic regulation of glycoconjugate biosynthesis because their expression on erythrocytes and in saliva has been thoroughly investigated in terms of classical genetics and the chemical structures and pathways for the formation of the antigens are now well understood. The primary protein products of the blood group genes are believed to be the glycosyltransferase enzymes that complete the biosynthesis of the determinants. The important controlling factors still to be elucidated are the genetic and environmental influences leading to the tissue specific expression of these antigens. The 3 types of regulation mechanisms discussed in this review are those arising: 1) from the specificity requirements of the glycosyltransferases encoded by the blood group genes; 2) from the competition or co-operation of glycosyltransferases encoded by genes at the same or independent loci; and 3) from the existence and tissue distribution of glycosyltransferases with related, but not identical, substrate specificities.     

Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry

Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.