Extracellular polysaccharides and glycoproteins of pathogenic bacteria assist in adherence, autoaggregation, biofilm formation, and host immune system evasion. As a result, considerable research in the field of glycobiology is dedicated to study the composition and function of glycans associated with virulence, as well as the enzymes involved in their biosynthesis with the aim to identify novel antibiotic targets. Especially, insights into the enzyme mechanism, substrate binding, and transition-state structures are valuable as a starting point for rational inhibitor design. An intriguing aspect of enzymes that generate or process polysaccharides and glycoproteins is the level of processivity. The existence of enzymatic processivity reflects the need for regulation of the final glycan/glycoprotein length and structure, depending on the role they perform. In this Review, we describe the currently reported examples of various processive enzymes involved in polymerization and transfer of sugar moieties, predominantly in bacterial pathogens, with a focus on the biochemical methods, to showcase the importance of studying processivity for understanding the mechanism.

We are interested in identifying and characterizing small molecule inhibitors of bacterial virulence factors for their potential use as anti-virulence inhibitors. We identified from high-throughput screening assays a potential activity for avasimibe, a previously characterized acyl-coenzyme A: cholesterol acyltransferase inhibitor, in inhibiting the NleB and SseK arginine glycosyltransferases from Escherichia coli and Salmonella enterica, respectively. Avasimibe inhibited the activity of the Citrobacter rodentium NleB, E. coli NleB1, and S. enterica SseK1 enzymes, without affecting the activity of the human serine/threonine N-acetylglucosamine (O-GlcNAc) transferase. Avasimibe was not toxic to mammalian cells at up to 200 µM and was neither bacteriostatic nor bactericidal at concentrations of up to 125 µM. Doses of 10 µM avasimibe were sufficient to reduce S. enterica abundance in RAW264.7 macrophage-like cells, and intraperitoneal injection of avasimibe significantly reduced C. rodentium survival in mice, regardless of whether the avasimibe was administered pre- or post-infection. We propose that avasimibe or related derivates created using synthetic chemistry may have utility in preventing or treating bacterial infections by inhibiting arginine glycosyltransferases that are important to virulence.

Glycosylation is the most frequent posttranslational modification of proteins and lipids influencing inter- and intracellular communication and cell adhesion. Altered glycosylation patterns are characteristically observed in tumour cells. Normal and altered carbohydrate chains are transferred to their acceptor structures via glycosyltransferases. Here, we present the correlation between the presence of three different glycosyltransferases and tumour characteristics.

Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen-Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.

The quantitative reverse-transcription polymerase chain reaction (RT-PCR) is a method used to quantify messenger RNA (mRNA) levels. It is particularly applicable to quantification of mRNAs that are transcribed at very low levels, such as glycosyltransferases (GTs). In this chapter, I describe preliminary experiments for obtaining conditions for a quantitative RT-PCR method to quantify transcript levels of GTs and related genes potentially involved in L-selectin-mediated lymphocyte homing in the mouse gastric mucosa infected by Helicobacter felis. This method was developed by modifying conventional RT-PCR protocols and does not require fluorescence-detecting thermal cyclers. The method described here is particularly useful for assaying large numbers of samples that require very accurate analysis.

Glycosyltransferases (GTs), the enzymes that catalyse glycosidic bond formation, create a diverse range of saccharides and glycoconjugates in nature. Understanding GTs at the molecular level, through structural and kinetic studies, is important for gaining insights into their function. In addition, this understanding can help identify those enzymes which are involved in diseases, or that could be engineered to synthesize biologically or medically relevant molecules. This review describes how structural data, obtained in the last 3-4 years, have contributed to our understanding of the mechanisms of action and specificity of GTs. Particular highlights include the structure of a bacterial oligosaccharyltransferase, which provides insights into N-linked glycosylation, the structure of the human O-GlcNAc transferase, and the structure of a bacterial integral membrane protein complex that catalyses the synthesis of cellulose, the most abundant organic molecule in the biosphere.

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.

Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti-tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC-subbranch with 160 GT sequences. We demonstrated that the YjiC-subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC-subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high-active GTs (Q66 and P77) but changed in two low-efficient GTs. Site-directed residues swapping at the two positions in the two low-active GTs (BssGT and BamGT) and the high-active GT BsGT-1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

Attachment of sugars to nitrogen and oxygen in peptides is ubiquitous in biology, but glycosylation of sulfur atoms has only been recently described. Here, we characterize two S-glycosyltransferases SunS and ThuS that selectively glycosylate one of five Cys residues in their substrate peptides; substitution of this Cys with Ser results in a strong decrease in glycosylation activity. Crystal structures of SunS and ThuS in complex with UDP-glucose or a derivative reveal an unusual architecture in which a glycosyltransferase type A (GTA) fold is decorated with additional domains to support homodimerization. Dimer formation creates an extended cavity for the substrate peptide, drawing functional analogy with O-glycosyltransferases involved in cell wall biosynthesis. This extended cavity contains a sharp bend that may explain the site selectivity of the glycosylation because the target Cys is in a Gly-rich stretch that can accommodate the bend. These studies establish a molecular framework for understanding the unusual S-glycosyltransferases.

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

Andrographis paniculata is an important traditional medicinal herb in South and Southeast Asian countries with diverse pharmacological activities that contains various flavonoids and flavonoid glycosides. Glycosylation can transform aglycones into more stable, biologically active and structurally diverse glycosides. Here, we report three glycosyltransferases from the leaves of A. paniculata (ApUFGTs) that presented wide substrate spectra for flavonoid glycosylation and exhibited multi-site glycosylation on the substrate molecules. They acted on the 7-OH position of the A ring and were able to glycosylate several other different types of compounds. The biochemical properties and phylogenetic analysis of these glycosyltransferases were also investigated. This study provides a basis for further research on the cloning of genes involved in glycosylation from A. paniculata and offers opportunities for enhancing flavonoid glycoside production in heterologous hosts. These enzymes are expected to become effective tools for drug discovery and for the biosynthesis of derivatives via flavonoid glycosylation.

The carbohydrate active enzyme (CAZy) database is an invaluable resource for glycobiology and currently contains 45 glycosyltransferase families that are represented in plants. Glycosyltransferases (GTs) have many functions in plants, but the majority are likely to be involved in biosynthesis of polysaccharides and glycoproteins in the plant cell wall. Bioinformatic approaches and structural modeling suggest that a number of protein families in plants include GTs that have not yet been identified as such and are therefore not included in CAZy. These families include proteins with domain of unknown function (DUF) DUF23, DUF246, and DUF266. The evidence for these proteins being GTs and their possible roles in cell wall biosynthesis is discussed.

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and β3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/β3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.

Plant hormones cytokinins (CKs) are one of the major mediators of physiological responses throughout plant life span. Therefore, a proper homeostasis is maintained by regulation of their active levels. Besides degradation, CKs are deactivated by uridine diphosphate glycosyltransferases (UGTs). Physiologically, CKs active levels decline in senescing organs, providing a signal to nutrients that a shift to reproductive tissues has begun. In this work, we show CK glucosides distribution in Arabidopsis leaves during major developmental transition phases. Besides continuous accumulation of N-glucosides we detected sharp maximum of the glucosides in senescence. This is caused prevalently by N7-glucosides followed by N9-glucosides and specifically also by trans-zeatin-O-glucoside (tZOG). Interestingly, we observed a similar trend in response to exogenously applied CK. In Arabidopsis, only three UGTs deactivate CKs in vivo: UGT76C1, UGT76C2 and UGT85A1. We thereby show that UGT85A1 is specifically expressed in senescent leaves whereas UGT76C2 is activated rapidly in response to exogenously applied CK. To shed more light on the UGTs physiological roles, we performed a comparative study on UGTs loss-of-function mutants, characterizing a true ugt85a1-1 loss-of-function mutant for the first time. Although no altered phenotype was detected under standard condition we observed reduced chlorophyll degradation with increased anthocyanin accumulation in our experiment on detached leaves accompanied by senescence and stress related genes modulated expression. Among the mutants, ugt76c2 possessed extremely diminished CK N-glucosides levels whereas ugt76c1 showed some specificity toward cis-zeatin (cZ). Besides tZOG, a broader range of CK glucosides was decreased in ugt85a1-1. Performing CK metabolism gene expression profiling, we revealed that activation of CK degradation pathway serves as a general regulatory mechanism of disturbed CK homeostasis followed by decreased CK signaling in all UGT mutants. In contrast, a specific regulation of CKX7, CKX1 and CKX2 was observed for each individual UGT mutant isoform after exogenous CK uptake. Employing an in silico prediction we proposed cytosolic localization of UGT76C1 and UGT76C2, that we further confirmed by GFP tagging of UGT76C2. Integrating all the results, we therefore hypothesize that UGTs possess different physiological roles in Arabidopsis and serve as a fine-tuning mechanism of active CK levels in cytosol.

The process of glycosylation has been studied extensively in prokaryotes but many questions still remain unanswered. Glycosyltransferase is the enzyme which mediates glycosylation and has its preference for the target glycosylation sites as well as for the type of glycosylation i.e. N-linked and O-linked glycosylation. In this study we carried out the bioinformatics analysis of one of the key enzymes of pgl locus from Campylobacter jejuni, known as PglB, which is distributed widely in bacteria and AglB from archaea. Relatively little sequence similarity was observed in the archaeal AglB(s) as compared to those of the bacterial PglB(s). In addition we tried to the answer the question of as to why not all the sequins Asp-X-Ser/Thr have an equal opportunity to be glycosylated by looking at the influence of the neighboring amino acids but no significant conserved pattern of the flanking sites could be identified. The software tool was developed to predict the potential glycosylation sites in autotransporter protein, the virulence factors of gram negative bacteria, and our results revealed that the frequency of glycosylation sites was higher in adhesins (a subclass of autotransporters) relative to the other classes of autotransporters.

Bacterial interactions with the environment- and/or host largely depend on the bacterial glycome. The specificities of a bacterial glycome are largely determined by glycosyltransferases (GTs), the enzymes involved in transferring sugar moieties from an activated donor to a specific substrate. Of these GTs their coding regions, but mainly also their substrate specificity are still largely unannotated as most sequence-based annotation flows suffer from the lack of characterized sequence motifs that can aid in the prediction of the substrate specificity.

Glycosyltransferases (GTs) are prevalent across the tree of life and regulate nearly all aspects of cellular functions. The evolutionary basis for their complex and diverse modes of catalytic functions remain enigmatic. Here, based on deep mining of over half million GT-A fold sequences, we define a minimal core component shared among functionally diverse enzymes. We find that variations in the common core and emergence of hypervariable loops extending from the core contributed to GT-A diversity. We provide a phylogenetic framework relating diverse GT-A fold families for the first time and show that inverting and retaining mechanisms emerged multiple times independently during evolution. Using evolutionary information encoded in primary sequences, we trained a machine learning classifier to predict donor specificity with nearly 90% accuracy and deployed it for the annotation of understudied GTs. Our studies provide an evolutionary framework for investigating complex relationships connecting GT-A fold sequence, structure, function and regulation.

Glycosylation is an important aspect of epigenetic regulation. Glycosyltransferase is a key enzyme in the biosynthesis of glycans, which glycosylates more than half of all proteins in eukaryotes and is involved in a wide range of biological processes. It has been suggested previously that homooligomerization in glycosyltransferases and other proteins might be crucial for their function. In this study, we explore functional homooligomeric states of glycosyltransferases in various organisms, trace their evolution, and perform comparative analyses to find structural features that can mediate or disrupt the formation of different homooligomers. First, we make a structure-based classification of the diverse superfamily of glycosyltransferases and confirm that the majority of the structures are indeed clustered into the GT-A or GT-B folds. We find that homooligomeric glycosyltransferases appear to be as ancient as monomeric glycosyltransferases and go back in evolution to the last universal common ancestor (LUCA). Moreover, we show that interface residues have significant bias to be gapped out or unaligned in the monomers, implying that they might represent features crucial for oligomer formation. Structural analysis of these features reveals that the majority of them represent loops, terminal regions, and helices, indicating that these secondary-structure elements mediate the formation of glycosyltransferases' homooligomers and directly contribute to the specific binding. We also observe relatively short protein regions that disrupt the homodimer interactions, although such cases are rare. These results suggest that relatively small structural changes in the nonconserved regions may contribute to the formation of different functional oligomeric states and might be important in regulation of enzyme activity through homooligomerization.