Specific Glycosyltransferases Research Articles

Stepwise Synthesis of Quercetin Bisglycosides Using Engineered Escherichia coli.

Synthesis of flavonoid glycoside is difficult due to diverse hydroxy groups in flavonoids and sugars. As such, enzymatic synthesis or biotransformation is an approach to solve this problem. In this report, we used stepwise biotransformation to synthesize two quercetin bisglycosides (quercetin 3-O-glucuronic acid 7-O-rhamnoside [Q-GR] and quercetin 3-O-arabinose-7-O-rhamnoside [Q-AR]) because quercetin O-rhamnosides contain antiviral activity. Two sequential enzymatic reactions were required to synthesize these flavonoid glycosides. We first synthesized quercetin 3-O-glucuronic acid [Q-G], and quercetin 3-O-arabinose-[Q-A] from quercetin using E. coli harboring specific uridine diphopsphate glycosyltransferase (UGT) and genes for UDP-glucuronic acid and UDP-arabinose, respectively. With each quercetin 3-O-glycoside, rhamnosylation using E. coli harboring UGT and the gene for UDP-rhamnose was conducted. This approach resulted in the production of 44.8 mg/l Q-GR and 45.1 mg/l Q-AR. This stepwise synthesis could be applicable to synthesize various natural product derivatives in case that the final yield of product was low due to the multistep reaction in one cell or when sequential synthesis is necessary in order to reduce the synthesis of byproducts.

Identification and characterization of the 4-epimerase AglW from the archaeon Methanococcus maripaludis.

Archaea are ubiquitous single-cell microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis, a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with N-linked tetrasaccharides that are essential for the proper assembly and function of archaella. The aglW gene, encoding the putative 4-epimerase AglW, plays a key role in the synthesis of the tetrasaccharide. The goal of our work was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop assays to determine its substrate specificity and properties. We carried out assays using UDP-Galactose, UDP-Glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and N-acetylglucosamine/N-acetylgalactosamine-diphosphate - lipid as substrates, coupled with specific glycosyltransferases. We showed that AglW has a broad specificity towards UDP-sugars and that Tyr151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The glycosyltransferase-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases.

Molecular Elucidation of Two Novel Seed Specific Flavonoid Glycosyltransferases in Soybean

Flavonoids are specialized plant secondary metabolites that are mainly present as glycoconjugates and function as attractants to pollinators and symbionts, UV protectants, allelochemicals, and have antimicrobial and antiherbivore activity for plant health. Because of the heterogeneity of UDP-glycosyltransferases (UGTs) for glycosylation in plants, their function in flavonoid glycosylation remains largely unknown in soybean and other legumes, particularly that of the UGT92 genes. Here, we identified 152 putative UGT92 genes across 48 plant species and elucidated their mode of duplication, expansion/deletion pattern, alignment, phylogenetic analysis, and genome-wide distribution. Two novel UGT-encoding genes Glyma14g04790 (UGT92G1) and Glyma15g03670 (UGT92G2) were isolated from soybean and their heterologous expression was optimized in Escherichia. coli. Both genes exhibited catalytic activity toward quercetin, kaempferol, and myricetin, with UDPglucose as the sugar donor and were characterized as flavanol-specific UGTs. High expression of both UGTs was observed in adaxial and abaxial parenchyma, suspensor cells, and adaxial and abaxial epidermis cells during seed development, suggesting that they are seed-specific flavanol glycosyltransferases in soybean. Co-expression analysis of UGT92 genes with their first and second neighborhood genes provided a basis for their network elucidation in soybean. We provide valuable information on the role of UGT92 in seed development via the glycosylation of multiple flavanols and the potential metabolic engineering of flavonoid compounds in both plants and E. coli.

Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for Enzymatic Bioorthogonal Labeling

In recent years, the development of the enzymatic bioorthogonal labeling strategy has offered exciting possibilities in the probing of structure-defined glycan epitopes. This strategy takes advantage of relaxed donor specificity and strict acceptor specificity of glycosyltransferases to label target glycan epitopes with bioorthogonal reactive groups carried by unnatural nucleotide sugars in vitro. The subsequent covalent conjugation by bioorthogonal chemical reactions with either fluorescent or affinity tags allows further visualization, quantification, or enrichment of target glycan epitopes. However, the application and development of the enzymatic labeling strategy have been hindered due to the limited availability of unnatural nucleotide sugars. Herein, a platform that combines chemical synthesis and enzymatic synthesis for the facile preparation of unnatural nucleotide sugars modified with diverse bioorthogonal reactive groups is described. By this platform, a total of 25 UDP-GlcNAc and UDP-GalNAc de...

Abstract 1840: Glycosylation of HER2+ human breast cancer cells control sensitivity to trastuzumab

Abstract Galectins decode glycan-containing information in a number of cell receptors adjusting signaling thresholds and modulating cellular functions. Upon specific binding to cell surface glycans these lectins control receptor clustering, endocytosis and signaling, influencing many physiological and pathological conditions. In particular, galectin-1 (Gal1), a 14kD prototype galectin, binds to terminal N-acetyllactosamine residues on glycosylated proteins in the absence of α2-6 sialic acid capping (Gal1 permissive glycophenotype). In early studies, our group demonstrated that tumors usurp the Gal1 pathway to evade immune surveillance and to promote aberrant angiogenesis, influencing sensitivity to anti-VEGF therapy. Here we aim to explore the glycosylation signature of HER2+ breast cancer cells and to investigate the effects of tumor-derived Gal1 on RTK activation and resistance to trastuzumab (TZ). We first selected three HER2+ human breast cancer cell lines with different response to TZ: JIMT-1 (resistant-TZR), BT-474 and SK-BR-3 (sensitive-TZS). To identify specific glycan structures, we used a panel of biotinylated plant lectins with different carbohydrate specificities. We found that TZS cell lines exhibited a Gal-1 restrictive glycophenotype characterized by high α2,6 sialic acid capping. Profiling of N-type glycans (WAX-HPLC) confirmed that TZSs cell lines exhibit higher frequency of α2,6 sialic acid residues compared to TZR JIMT-1. This effect was further substantiated by RT-PCR analysis of specific glycosyltransferases responsible of Gal1 ligands biosynthesis. These results were supported by analysis of raw data from public databases arrays (GSE62327) showing that patients who presented complete response to TZ exhibited higher levels of ST6GAL1, a glycosyltransferase responsible for α2-6 sialic acid capping. In accordance with the glycophenotype, TZR cell line bound and expressed higher levels of Gal1 when compared to TZS cell lines by Western blot, RT-PCR and ELISA of conditioned medium, suggesting a positive autocrine loop that could modulate cell behavior. Moreover, in silico analysis of raw data from the Long HER Study (GSE44272) revealed that patients with poor response to TZ expressed higher levels of Gal1 mRNA than long-term responders, reinforcing our hypothesis from a clinical standpoint. Finally, in order to elucidate Gal1 implications in TZ resistance, we knocked down Gal1 in JIMT-1 cell line using shRNA strategies. Remarkably, absence of Gal1 expression sensitized JIMT-1 cells to TZ-inhibition in vitro, and the resistant phenotype was further reestablished by addition of human recombinant Gal1. In summary, our study suggests that individual HER2+ human breast cancer cells display particular “glycosylation signatures” which, in association with Gal1 expression pattern, may control resistance to anti-HER2 targeted therapy and may predict breast cancer clinical outcome. Citation Format: Ramiro M. Perrotta, Tomás Dalotto-Moreno, Alejandro Cagnoni, Karina Mariño, Gabriel Rabinovich, Mariana Salatino. Glycosylation of HER2+ human breast cancer cells control sensitivity to trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1840.

Heterologous expression of Shigella dysenteriae serotype 1 O-antigen analog in Escherichia coli K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster. The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached. Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

Disseminated castration-resistant prostate cancer (CRPC) is a common disease in men that is characterized by limited survival and resistance to androgen-deprivation therapy. The increase in human epidermal growth factor receptor 2 (HER2) signaling contributes to androgen receptor activity in a subset of patients with CRPC; however, enigmatically, HER2-targeted therapies have demonstrated a lack of efficacy in patients with CRPC. Aberrant glycosylation is a hallmark of cancer and involves key processes that support cancer progression. Using transcriptomic analysis of prostate cancer data sets, histopathologic examination of clinical specimens, and in vivo experiments of xenograft models, we reveal in this study a coordinated increase in glycan-binding protein, galectin-4, specific glycosyltransferases of core 1 synthase, glycoprotein- N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) and ST3 beta-galactoside α-2,3-sialyltransferase 1 (ST3GAL1), and resulting mucin-type O-glycans during the progression of CRPC. Furthermore, galectin-4 engaged with C1GALT1-dependent O-glycans to promote castration resistance and metastasis by activating receptor tyrosine kinase signaling and cancer cell stemness properties mediated by SRY-box 9 (SOX9). This galectin-glycan interaction up-regulated the MYC-dependent expression of C1GALT1 and ST3GAL1, which altered cellular mucin-type O-glycosylation to allow for galectin-4 binding. In clinical prostate cancer, high-level expression of C1GALT1 and galectin-4 together predict poor overall survival compared with low-level expression of C1GALT1 and galectin-4. In summary, MYC regulates abnormal O-glycosylation, thus priming cells for binding to galectin-4 and downstream signaling, which promotes castration resistance and metastasis.-Tzeng, S.-F., Tsai, C.-H., Chao, T.-K., Chou, Y.-C., Yang, Y.-C., Tsai, M.-H., Cha, T.-L., Hsiao, P.-W. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

Ectopic expression of UGT84A2 delayed flowering by indole-3-butyric acid-mediated transcriptional repression of ARF6 and ARF8 genes in Arabidopsis.

Ectopic expression of auxin glycosyltransferase UGT84A2 in Arabidopsis can delay flowering through increased indole-3-butyric acid and suppressed transcription of ARF6, ARF8 and flowering-related genes FT, SOC1, AP1 and LFY. Auxins are critical regulators for plant growth and developmental processes. Auxin homeostasis is thus an important issue for plant biology. Here, we identified an indole-3-butyric acid (IBA)-specific glycosyltransferase, UGT84A2, and characterized its role in Arabidopsis flowering development. UGT84A2 could catalyze the glycosylation of IBA, but not indole-3-acetic acid (IAA). UGT84A2 transcription expression was clearly induced by IBA. When ectopically expressing in Arabidopsis, UGT84A2 caused obvious delay in flowering. Correspondingly, the increase of IBA level, the down-regulation of AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, and the down-regulation of flowering-related genes such as FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1), APETALA1 (AP1), and LEAFY(LFY) were observed in transgenic plants. When exogenously applying IBA to wild-type plants, the late flowering phenotype, the down-regulation of ARF6, ARF8 and flowering-related genes recurred. We examined the arf6arf8 double mutants and found that the expression of flowering-related genes was also substantially decreased in these mutants. Together, our results suggest that glycosyltransferase UGT84A2 may be involved in flowering regulation through indole-3-butyric acid-mediated transcriptional repression of ARF6, ARF8 and downstream flowering pathway genes.

Abstract 5801: Loss of core 1 β1,3-galactosyltransferase (C1GALT1) in pancreatic cancer leads to altered mucin glycosylation and increased tumor aggressiveness

Abstract Background: Aberrant expression of mucins underlie pancreatic cancer (PC) progression and metastasis. Mucin-type O-glycosylation is the principal post-translational modification on mucins and is regulated by myriad of glycosyltransferases. Not much has been investigated about the specific glycosyltransferases involved in increased PC aggressiveness. We found that core 1 β1,3-galactosyltransferase (C1GALT1), which is essential for O-glycosylation, is not expressed in some of the PC patients. C1GALT1 adds galactose in O-glycosylation pathway that forms core 1 structure, which is usually elongated to several carbohydrate structures. Loss of C1GALT1 is accompanied by incomplete O-glycosylation that results in increased expression of truncated carbohydrate antigens (Tn and sTn). Increased expression of these truncated carbohydrate antigens on mucins has been associated with augmented aggressiveness in several malignancies. Based on these results, we hypothesized that loss of C1GALT1 in PC patients is associated with altered mucin O-glycosylation and increased aggressiveness. Methods: Expression of C1GALT1 was examined in HPNE (human pancreatic nestin expressing cells) and oncogenic transformed HPNE cells. To understand the role of C1GALT1 in PC, CRISPR/Cas9 mediated knockout of CIGALT1 was performed in PC cells. Impact of C1GALT1 knockout on glycosylation profile and mucin glycosylation was evaluated using lectin blotting and lectin-pull down assay. Wound-healing assay was performed to examine the effect of C1GALT1 knockout on migration. To gain mechanistic insight, protein expression of signaling molecules involved in growth and motility was evaluated using western blotting. Orthotopic implantation of C1GALT1 knockout cells was carried in pancreas of nude mice to study its role in tumor growth and metastasis. KPC mouse model (KrasG12D; Trp53R172H/+; Pdx-1- Cre) of PC progression was crossed with C1galt1 floxed mice (resulted in KPCC mice) to study the involvement of C1galt1 in disease progression and metastasis in vivo. Results: Oncogenic transformed HPNE cells showed decreased expression of C1GALT1 as compared to untransformed HPNE cells. Knockout of C1GALT1 in PC cells resulted in increased expression of truncated carbohydrate antigens (Tn and sTn). Further, knockout cells displayed increased cellular protrusions, which was associated with significantly increased migration. Western blotting demonstrated increased expression of proteins involved in growth and motility. Lectin-pull down assay revealed altered MUC16 glycosylation in C1GALT1 knockout cells. In vivo studies using orthotopic implantation and KPCC mouse model demonstrated increased tumor weight and metastasis with knockout of C1GALT1. Conclusion: Overall, our results indicate that loss of C1GALT1 in PC is associated with altered MUC16 glycosylation and increased PC aggressiveness. Citation Format: Seema Chugh, Satyanarayana Rachagani, Xinheng Yu, Sriram Neelamegham, Lijun Xia, Michel M. Ouellette, Moorthy P. Ponnusamy, Surinder K. Batra. Loss of core 1 β1,3-galactosyltransferase (C1GALT1) in pancreatic cancer leads to altered mucin glycosylation and increased tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5801. doi:10.1158/1538-7445.AM2017-5801

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

ABSTRACTDespite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc‐glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N‐linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N‐linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc‐glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570–1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

FUT2 genetic variants as predictors of tumor development with hepatocellular carcinoma.

Lewis antigens related to the ABO blood group are fucosylated oligosaccharides and are synthesized by specific glycosyltransferases (FUTs). FUTs are involved in various biological processes including cell adhesion and tumor progression. The fucosyltransferase-2 gene (FUT2) encodes alpha (1,2) fucosyltransferase, which is responsible for the addition of the alpha (1,2)-linkage of fucose to glycans. Aberrant fucosylation occurs frequently during the development and progression of hepatocellular carcinoma (HCC). However, the association of FUT2 polymorphisms with HCC development has not been studied. Therefore, we aimed to investigate the association of FUT2 polymorphisms with demographic, etiological, and clinical characteristics and with susceptibility to HCC. In this study, a total of 339 patients and 720 controls were recruited. The genotypes of FUT2 at four single-nucleotide polymorphisms (SNPs; rs281377, rs1047781, rs601338, and rs602662) were detected by real-time polymerase chain reaction from these samples. Compared with the wild-type genotype at SNP rs1047781, which is homozygous for nucleotides AA, at least one polymorphic T allele (AT or TT) displayed significant association with clinical stage (p = 0.048) and tumor size (p = 0.022). Our study strongly implicates the polymorphic locus rs1047781 of FUT2 as being associated with HCC development.

Labeling glycans on living cells by a chemoenzymatic glycoengineering approach.

ABSTRACTStructural glycobiology has traditionally been a challenging field due to a limited set of tools available to investigate the diverse and complex glycan molecules. However, we cannot ignore that glycans play critical roles in health as well as in disease, and are present in more than 50% of all proteins and on over 80% of all surface proteins. Chemoenzymatic glycoengineering (CGE) methods are a powerful set of tools to synthesize complex glycans, but the full potential of these methods have not been explored in cell biology yet. Herein, we report the labeling of live Chinese hamster ovary (CHO) cells by employing three highly specific glycosyltransferases: a sialyltransferase, a galactosyltransferase, and an N-acetyl-glucosaminyl transferase. We verified our results by bio-orthogonal blots and further rationalized them by computational modeling. We expect CGE applications in cell biology to rise and their implementation will assist in structural-functional discoveries in glycobiology. This research will contribute to this effort.

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications.

A major branch of glycobiology and glycan-focused biomedicine studies the interaction between carbohydrates and other biopolymers, most importantly, glycan-binding proteins. Today, this research into glycan-biopolymer interaction is unthinkable without glycan arrays, tools that enable high-throughput analysis of carbohydrate interaction partners. Glycan arrays offer many applications in basic biochemical research, for example, defining the specificity of glycosyltransferases and lectins such as immune receptors. Biomedical applications include the characterization and surveillance of influenza strains, identification of biomarkers for cancer and infection, and profiling of immune responses to vaccines. Here, we review major applications of glycan arrays both in basic and applied research. Given the dynamic nature of this rapidly developing field, we focus on recent findings.

Mycoplasma agalactiae Secretion of β-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation.

Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear β-(1→6)-glucopyranose structure [β-(1→6)-glucan]. Secretion of β-(1→6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of β-(1→6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, β-(1→6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3(T), another pathogen of small ruminants. Many if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular β-(1→6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached β-(1→6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.

Regulation of O-antigen biosynthesis in Yersinia enterocolitica.

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The LPS molecule consists of three regions: (i) the lipid A, a polar glycolipid in which a disaccharide backbone is replaced with six or seven fatty acid; (ii) the core, an oligosaccharide often rich in negatively charged groups and, (iii) the O-antigen, a polysaccharide that protrudes into the surroundings. It is widely known that the lipid A part is responsible for most of the biological effects of LPS; however, there is increasing body of evidence showing that O-antigen modulates the biological activity of lipid A and also plays a critical role in the initial interaction of bacterium with the host. The biosynthesis of heteropolymeric O-antigens whose O-units are composed of different sugar residues starts in the cytoplasm by the activation of sugar 1-phosphates in a reaction with one of the nucleoside triphosphates, followed by different biosynthetic pathways to give rise to cytoplasmically located individual nucleoside diphosphate (NDP)-activated sugar precursors. The assembly of the O-units takes place on the cytoplasmic face of the inner membrane and involves the transfer of sugar residues from the NDP-sugar precursors onto a lipid carrier molecule, undecaprenylphosphate (Und-P), by specific glycosyltransferases. The completed O-units still assembled on the Und-P are translocated by the O-unit flippase, Wzx, across the inner membrane to the periplasmic face. There the O-units are polymerized by the O-antigen polymerase, Wzy, into an O-antigen that is ligated to the lipid Acore structure by the O-antigen ligase encoded by the waaL gene of the core

Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes

Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha-treated adipocytes correlated with the regulation of specific glycosyltransferases, including the up-regulation of B4GalT5 and Ggta1 galactosyltransferases and down-regulation of ST3Gal6 sialyltransferase. Knockdown of B4GalT5 down-regulated the terminal di-galactose N-glycans, confirming the involvement of this enzyme in the TNF-alpha-regulated N-glycome. SILAC-based quantitative glycoproteomics of enriched N-glycopeptides with and without deglycosylation were used to identify the protein and glycosylation sites modified with these regulated N-glycans. The combined proteome and glycoproteome workflow provided a relative quantification of changes in protein abundance versus N-glycosylation occupancy versus site-specific N-glycans on a proteome-wide level. This revealed the modulation of N-glycosylation on specific proteins in IR, including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane.

Abstract 4983: GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer

Abstract Purpose: MUC4 is one of the membrane-bound mucins that is expressed de novo during pancreatic cancer (PC) and contributes to pathogenesis. Further, MUC4 is aberrantly glycosylated in PC and some of its tumor-promoting effects are, in part, mediated by interactions facilitated by the glycan epitopes, which are generally masked under normal conditions. Mucins predominantly undergo O-glycosylation that is initiated by a family of enzymes known as GALNTs (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase), catalyzing the transfer of first sugar residue (GalNAc) to ser/thr residues in mucin tandem repeat domains. Not much is known about the specific glycosyltransferases (GTs) involved in MUC4 glycosylation. Understanding the specific enzymes regulating differential glycosylation of MUC4 in cancer may be important for developing therapeutic strategies for modulating MUC4 function in PC. Thus, the goal of this study was to identify specific GALNT(s) catalyzing MUC4 glycosylation and investigate its role in differential MUC4 glycosylation in PC. Materials & Methods: PCR array analysis was performed in a panel of MUC4 expressing and non-expressing PC cell lines, normal pancreatic cancer cells and MUC4 expressing normal colon cells to identify differentially expressed GTs. To determine the altered glycosylation of MUC4 in normal versus cancerous conditions, Co-immunoprecipitation (Co-IP) studies were conducted using several carbohydrate-specific antibodies. Stable knockdown of GALNT3 was performed in PC and normal colon cells to understand its involvement in differential MUC4 glycosylation. To determine the functional implications of GALNT3 mediated aberrant glycosylation, various in vitro assays such as colony formation and migration were carried out. Results: PCR array and immunoblot analysis demonstrated GALNT3 as one of the GTs upregulated in MUC4 expressing cells as compared to MUC4 non-expressing PC cells. Interestingly, Co-IP analysis revealed differential expression of carbohydrates epitopes such as STn, SLex, SLea on MUC4 in PC and normal colon cells. GALNT3 knockdown in cancer cells resulted in decreased expression of Tn but simultaneous increase in STn epitope. Interestingly, we observed significant increase in colony formation and migration in GALNT3 knockdown cells. This was associated with increase in proliferation and EMT markers in GALNT3 knockdown PC cells such as N-cadherin, vimentin and phosphorylated ErbBs. Conclusion: Overall, our results suggested a correlation of MUC4 and GALNT3 expression, and knockdown of GALNT3 resulted in altered MUC4 glycosylation rather than complete abrogation, suggesting a possible compensatory increase in other GALNTs and other GTs. Further investigations along these lines will provide insight into specific GTs regulating MUC4 glycosylation and glycan epitope-mediated MUC4 functions in cancer. Citation Format: Seema Chugh, Vinayaga Srinivasan Gnanapragassam, Maneesh Jain, Moorthy Ponnusamy, Surinder Kumar Batra. GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2015-4983

Ginsenoside Rg3 suppresses FUT4 expression through inhibiting NF-κB/p65 signaling pathway to promote melanoma cell death.

Abnormal glycosylation is catalyzed by the specific glycosyltransferases and correlates with tumor cell apoptosis. Increased fucosyltransferase IV (FUT4) is seen in many types of cancer, and manipulating FUT4 expression through specific signaling pathway inhibits cell growth and induces apoptosis. NF-κB is known playing a vital role to control cell growth and apoptosis. Ginsenoside Rg3 is an herbal medicine with strong antitumor activity through inhibiting tumor growth and promoting tumor cell death. However, whether Rg3-induced inhibition on tumor development involves reduced NF-κB signaling and FUT4 expression remains unknown. In the present study, we found that Rg3 suppressed FUT4 expression by abrogating the binding of NF-κB to FUT4 promoter through inhibiting the expression of signaling molecules of NF-κB pathway, reducing NF-κB DNA binding activity and NF-κB transcription activity. NF-κB inhibitor (Bay 11-7082) or knocking down p65 expression by p65 siRNA also led to a significant decreased FUT4 expression. In addition, Rg3 induced apoptosis by activating both extrinsic and intrinsic apoptotic pathways. Moreover, in a xenograft mouse model, Rg3 downregulated FUT4 and NF-κB/p65 expression and suppressed melanoma cell growth and induced apoptosis without any noticeable toxicity. In conclusion, Rg3 induces tumor cell apoptosis correlated with its inhibitory effect on NF-κB signaling pathway-mediated FUT4 expression. Results suggest Rg3 might be a novel therapy agent for melanoma treatment.