Glycosyltransferases as biocatalysts

Glycosyltransferases are useful synthetic tools for the preparation of natural oligosaccharides, glycoconjugates and their analogues. High expression levels of recombinant enzymes have allowed their use in multi-step reactions, on mg to multi-gram scales. Since glycosyltransferases are tolerant with respect to utilizing modified donors and acceptor substrates they can be used to prepare oligosaccharide analogues and for diversification of natural products. New sources of enzymes are continually discovered as genomes are sequenced and they are annotated in the Carbohydrate Active Enzyme (CAZy) glycosyltransferase database. Glycosyltransferase mutagenesis, domain swapping and metabolic pathway engineering to change reaction specificity and product diversification are increasingly successful due to advances in structure-function studies and high throughput screening methods.     

Mammalian glycosyltransferases: genomic organization and protein structure.

In recent years, several glycosyltransferase genes and cDNAs have been cloned and characterized. Although the glycosyltransferases seem to share the same general architecture, there is only little sequence similarity between the various enzymes. Moreover, a comparison of the organization of the genes shows that there is no common pattern of intron-exon structure. In addition, there seems to be little or no correlation between glycosyltransferase exons and protein domains. Taken together, these observations suggest that many of the glycosyltransferase genes evolved independently. So far, only two glycosyltransferase gene families have been described. These families may have evolved by exon-shuffling, or by gene duplication and subsequent divergence. For specific glycosyltransferases, mechanisms such as alternative splicing and alternative promoter usage play a role in the production of multiple protein isoforms from a single gene. These isoenzymes may differ in their enzymatic properties or cellular localization.     

Construction of a human glycogene library and comprehensive functional analysis

Eighteen years have passed after the first mammalian glycosyltransferase was cloned. At the beginning of April, 2001, 110 genes for human glycosyltransferases, including modifying enzymes for carbohydrate chains such as sulfotransferases, had been cloned and analyzed. We started the Glycogene Project (GG project) in April 2001, a comprehensive study on human glycogenes with the aid of bioinformatic technology. The term glycogene includes the genes for glycosyltransferases, sulfotransferases adding sulfate to carbohydrates and sugar-nucleotide transporters, etc. Firstly, as many novel genes, which are the candidates for glycogenes, as possible were searched using bioinformatic technology in databases. They were then cloned and expressed in various expression systems to detect the activity for carbohydrate synthesis. Their substrate specificity was determined using various acceptors.     

Biocatalytic synthesis of oligosaccharides

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.     

Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.     

Tetracycline-regulated overexpression of glycosyltransferases in Chinese hamster ovary cells.

The glycosylation patterns of recombinant therapeutic glycoproteins can be engineered by overexpression of glycosyltransferases in the host cells used for glycoprotein production. Most prior glycosylation engineering experiments have involved constitutive expression of cloned glycosyltransferases. Here we use tetracycline-regulated expression of two glycosyltransferases, N-acetylglucosaminlytransferases III and V (GnTIII and GnTV) to manipulate glycoform biosynthesis in Chinese hamster ovary (CHO) cells and to study the effect of glycosyltransferase overexpression on this host. The amount of GnTIII and GnTV in these cells, and the glycosylation patterns of several cellular glycoproteins, could be controlled simply by manipulating the concentration of tetracycline in the culture medium. Using this system, it was found that overexpression of either GnTIII or GnTV to high levels led to growth inhibition and was toxic to the cells, indicating that this may be a general feature of glycosyltransferase overexpression. This phenomenon has not been reported previously, probably due to the widespread use of constitutive promoters, and should be taken into account when designing vectors for glycosylation engineering. The growth inhibition effect sets an upper limit to the level of glycosyltransferase overexpression, and may thereby also limit the maximum extent of in vivo modification of poorly accessible glycosylation sites. Also, such inhibition implies a bound on constitutive glycosyltransferase expression which can be cloned.     

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.     

Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.     

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.     

Biochemical characterization of a glycosyltransferase homolog from an oral pathogen Fusobacterium nucleatum as a human glycan-modifying enzyme

Bacterial glycosyltransferases have drawn growing attention as economical enzymes for oligosaccharide synthesis, with their easy expression and relatively broad substrate specificity. Here, we characterized a glycosyltransferase homolog (Fnu_GT) from a human oral pathogen, Fusobacterium nucleatum. Bioinformatic analysis showed that Fnu_GT belongs to the glycosyltransferases family II. The recombinant Fnu_GT (rFnu_GT) expressed in Escherichia coli displayed the highest glycosylation activity when UDP-galactose (Gal) was used as a donor nucleotide-sugar with heptose or Nacetylglucosamine (GlcNAc) as an acceptor sugar. Interestingly, rFnu_GT transferred the galactose moiety of UDP-Gal to a nonreducing terminal GlcNAc attached to the trimannosyl core glycan, indicating its potential as an enzyme for humantype N-glycan synthesis.     

Histochemical and cytochemical localization of blood group antigens.

The oligosaccharide structures of blood group antigens are not the primary gene products; they are constructed in a stepwise manner by adding particular sugar to precursor oligosaccharides via several glycosyltransferases coded for by different blood group genes (Watkins 1966, 1978, 1980). Consequently, final profiles of antigens expressed in each cell type are influenced by many different factors such as the intrinsic composition of glycosyltransferase species which are defined by the genotype of the individuals, relative activity or amount of these enzymes (repression, derepression or induction of the enzymes), competition between enzymes with overlapping substrate specificity, the organization of the enzymes in membranes, utilizability of precursors and specific substrate sugars, and the activity level of degradating enzymes. Changes in the antigen profiles during maturation, differentiation and malignant transformation are thought to be intimately related to the variability of these factors. Although great importance attaches to histo- and cytochemical information on the distribution and levels of glycosyltransferases and messenger RNA corresponding to the relevant enzyme, detailed and precise localization of the blood group antigens and their variants is the base line for analyzing these complex factors. On the basis of individual genotype and histochemical findings about the antigen distribution and the interrelationship between cells and cellular components producing different antigenic structures (cellular and subcellular mosaicism), we can deduce precursor oligosaccharide levels as well as the status of gene activation and its primary product, glycosyltransferases. Thus, these findings are a prerequisite for further analysis at the molecular genetic level. As emphasized in this article, lectin staining or immunostaining methods with MAbs combined with glycosidase digestion procedures are powerful tools for in situ analysis of carbohydrate structures in histochemical systems. Although in some cases valuable results have been obtained by applying the technique, our knowledge concerning the distribution of complex carbohydrate structures is still far from satisfactory. Along with well defined MAbs and lectins, the key to developing our methods further is successful introduction of glycosidases, in particular, endoglycosidases since these reagents are indispensable for analyzing the inner core structures and glycoconjugate species of the blood group antigens. Application of these techniques at the ultrastructural level is an alluring possibility, even though many difficulties must be overcome. Although their functional roles have not yet been determined, a diverse array of macromolecules is known to be decorated with blood group-related antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transcriptome profiling of bovine milk oligosaccharide metabolism genes using RNA-sequencing

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk.     

Synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of O-antigen repeating units

O-antigens of Gram negative bacteria are polysaccharides covalently attached to lipopolysaccharides (LPS) that have roles as virulence factors. Due to the lack of defined substrates for in vitro assays only a few of the enzymes involved in the biosynthesis of O-antigens have been studied. Many O-antigens have GlcNAc at the reducing end of the oligosaccharide chain linked to pyrophosphate-lipid. We therefore designed and synthesized a series of GlcNAc-pyrophosphate-lipid analogs of the natural GlcNAc-pyrophosphate-undecaprenol acceptor substrate for studies of the acceptor specificities of O-antigen biosynthetic enzymes. We synthesized analogs with modifications of the pyrophosphate bond as well as the lipid chain. These compounds will be useful for the specificity studies of many bacterial glycosyltransferases. Knowledge of the substrate specificities is the basis for the development of specific glycosyltransferase inhibitors that could block O-antigen biosynthesis.     

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.     

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.     

Bioengineered bugs expressing oligosaccharide receptor mimics: toxin-binding probiotics for treatment and prevention of enteric infections

Many microbial pathogens recognize oligosaccharides displayed on the surface of host cells as receptors for toxins and adhesins. These ligand-receptor interactions are critical for disease pathogenesis, making them promising targets for novel anti-infectives. One strategy with particular utility against enteric infections involves expression of molecular mimics of host oligosaccharides on the surface of harmless bacteria capable of surviving in the gut. This can be achieved in Gram-negative bacteria by manipulating the outer core region of the lipopolysaccharide (LPS) through expression of cloned heterologous glycosyltransferases. The resultant chimeric LPS molecules are incorporated into the outer membrane by the normal assembly route and presented as a closely packed 2-D array of receptor mimics. Several such "designer probiotics" have been constructed, and these bind bacterial toxins in the gut lumen with very high avidity, blocking their uptake by host cells and thereby preventing disease.     

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.     

Towards understanding the functional role of the glycosyltransferases involved in the biosynthesis of Moraxella catarrhalis lipooligosaccharide

The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.     

Altered mRNA expression of glycosyltransferases in human gastric carcinomas.

Biosynthesis of carbohydrate structures is tissue-specific and developmentally regulated by glycosyltransferases like fucosyl-, sialyl- and N-acetylglucosaminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumor subpopulations with different adhesion properties. The aim of this contribution was to directly compare mRNA expression of several glycosyltransferases in surgical specimens of gastric carcinomas. Carcinoma specimens were classified and characterized according to the WHO/UICC system. In each case, the expression of 12 glycosyltransferase enzymes was studied simultaneously by RT-PCR. For semi-quantitative analysis, amplification of the sample sequence was compared with that of beta-actin, co-amplified within the same tube. Expression of N-acetylglucosaminyltransferase V in gastric carcinomas was significantly enhanced compared to normal tissue. Also, expression of sialyltransferase ST3Gal-IV and fucosyltransferase FT-IV was significantly enhanced in carcinoma tissue. No significant differences in glycosyltransferase expression were found in samples positive for Helicobacter pylori or between the different gastric regions. Thus, carcinogenesis is characterized by specific alterations in mRNA expression of several glycosyltransferases. Future studies will show whether RT-PCR detection of the expression of these enzymes could be helpful for prognostic purposes.     

Large-scale production of oligosaccharides using engineered bacteria

Rapid advances in the cloning and expression of glycosyltransferase genes, especially from bacteria, could open the way to overcoming difficulties in the mass production of oligosaccharides. The large-scale production of oligosaccharides using either glycosyltransferases isolated from engineered microorganisms or whole cells as an enzyme source could promote a new era in the field of carbohydrate synthesis.