Control of T Cell-mediated autoimmunity by metabolite flux to N-glycan biosynthesis

Autoimmunity is a complex trait disease where the environment influences susceptibility to disease by unclear mechanisms. T cell receptor clustering and signaling at the immune synapse, T cell proliferation, CTLA-4 endocytosis, T(H)1 differentiation, and autoimmunity are negatively regulated by beta1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. Beta1,6GlcNAc-branched N-glycan expression in T cells is dependent on metabolite supply to UDP-GlcNAc biosynthesis (hexosamine pathway) and in turn to Golgi N-acetylglucosaminyltransferases Mgat1, -2, -4, and -5. In Jurkat T cells, beta1,6GlcNAc-branching in N-glycans is stimulated by metabolites supplying the hexosamine pathway including glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not by control metabolites mannosamine, galactose, mannose, succinate, or pyruvate. Hexosamine supplementation in vitro and in vivo also increases beta1,6GlcNAc-branched N-glycans in na?ve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, T(H)1 differentiation, experimental autoimmune encephalomyelitis, and autoimmune diabetes in non-obese diabetic mice. Our results indicate that metabolite flux through the hexosamine and N-glycan pathways conditionally regulates autoimmunity by modulating multiple T cell functionalities downstream of beta1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A TOOLBOX FOR SEQUENTIAL PLANT N-GLYCAN PROCESSING*

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N₂M₃FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.     

N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway

Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5^−/− mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5^−/− cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5^−/− hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor.     

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc^min/+ mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc^min/+ mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.     

Genome-scale identification of UDP-GlcNAc-dependent pathways

Metabolite flux to UDP-GlcNAc and Golgi N-glycan biosynthesis regulates surface residency of glycoprotein receptors and transporters, and thus sensitivities of cells to extracellular cues. Salvage of GlcNAc increases UDP-GlcNAc and branching of N-glycans progressively, but displays an optimum for cell proliferation and bulk endocytosis in mouse NMuMG and human HEK293T epithelial cells. In this report, we measured global changes in gene expression in low and high GlcNAc-supplemented cells. Genes upregulated by high GlcNAc included the EGF and TGF-beta signaling pathways and cell cycle checkpoint, while downregulated genes indicated lower metabolic activity. Genes increased or decreased by high GlcNAc were assessed by transfecting cells with small interfering RNA (siRNA) and measuring effects on three phenotypes: proliferation and bulk endocytosis, and beta1,6GlcNAc-branching of N-glycans. siRNA targeting LGALS3, WBSCR17, PHF3, SDC2 and CTNNAL1 partially reversed the GlcNAc-induced phenotypes, suggesting a role for galectin-3/N-glycans, proteoglycans, O-glycans, and junctional cell adhesion.     

UDP-N-acetylglucosamine Transporter (SLC35A3) Regulates Biosynthesis of Highly Branched N-Glycans and Keratan Sulfate

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply β-1,3-N-acetylglucosaminyl-transferase 7 (β3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.     

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-D-glucosamine

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni²⁺ and Fe³⁺ have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)_x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)₄ oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co²⁺ and Ni²⁺ under aerobic conditions, and Co²⁺, Ni²⁺ and Fe²⁺ under anaerobic conditions, but decreased activity with Zn²⁺. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.     

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae: STRUCTURAL CHARACTERIZATION AND IN SITU SYNTHESIS*S?

Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (¹H and ¹³C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[¹⁴C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts contains two types of β-glucans. In the former yeast, branched β-(1,3)-glucan accounts for ∼50–55%, whereas β-(1,6)-glucan represents 10–15% of the total yeast cell wall polysaccharides, each chain of the latter extending up to 140–350 glucose residues in length. The amount of 3,6-branched glucose residues varies with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida albicans, and Schizosaccharomyces pombe, respectively (1). β-(1,6)-Glucan stabilizes the cell wall, since it plays a central role as a linker for specific cell wall components, including β-(1,3)-glucan, chitin, and mannoproteins (2, 3). However, the exact structure of the β-(1,6)-glucan and the mode of biosynthesis of this polymer are largely unknown. In S. pombe, immunodetection studies suggested that synthesis of this polymer backbone begins in the endoplasmic reticulum, with extension occurring in the Golgi (4) and final processing at the plasma membrane. In S. cerevisiae, Montijn and co-workers (5), by immunogold labeling, detected β-(1,6)-glucan at the plasma membrane, suggesting that the synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and their homologues, SKN1 and KNH1, have been reported to be involved in β-(1,6)-glucan synthesis in S. cerevisiae, C. albicans, and Candida glabrata (6–10). Among all of these genes, the ones that seem to play the major synthetic role are KRE5 and KRE9, since their disruption caused significant reduction (100 and 80%, respectively, relative to wild type) in the cell wall β-(1,6)-glucan content (11–13).To date, the biochemical reaction responsible for the synthesis of β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in most cases, when membrane preparations are incubated with UDP-glucose, only linear β-(1,3)-glucan polymers are produced, although some studies have reported the production of low amounts of β-(1,6)-glucans by membrane preparations (14–17). These data suggest that disruption of the fungal cell prevents or at least has a strong negative effect on β-(1,6)-glucan synthesis. The use of permeabilized cells, which allows substrates, such as nucleotide sugar precursors, to be readily transported across the plasma membrane, is an alternative method to study in situ cell wall enzyme activities (18–22). A number of methods have been developed to permeabilize the yeast cell wall (23), of which osmotic shock was successfully used to demonstrate β-(1,3)-glucan and chitin synthase activities (20, 24). Herein, we describe the biochemical activity responsible for β-(1,6)-glucan synthesis using permeabilized S. cerevisiae cells and UDP-[¹⁴C]glucose as a substrate. We also have analyzed the physicochemical parameters of this activity and chemically characterized the end product and its structural organization within the mature yeast cell wall.     

A Bifunctional Enzyme in a Single Gene Catalyzes the Incorporation of GlcN into the Aeromonas Core Lipopolysaccharide

The core lipopolysaccharide (LPS) of Aeromonas hydrophila AH-3 and Aeromonas salmonicida A450 is characterized by the presence of the pentasaccharide α-d-GlcN-(1→7)-l-α-d-Hep-(1→2)-l-α-d-Hep-(1→3)-l-α-d-Hep-(1→5)-α-Kdo. Previously it has been suggested that the WahA protein is involved in the incorporation of GlcN residue to outer core LPS. The WahA protein contains two domains: a glycosyltransferase and a carbohydrate esterase. In this work we demonstrate that the independent expression of the WahA glycosyltransferase domain catalyzes the incorporation of GlcNAc from UDP-GlcNAc to the outer core LPS. Independent expression of the carbohydrate esterase domain leads to the deacetylation of the GlcNAc residue to GlcN. Thus, the WahA is the first described bifunctional glycosyltransferase enzyme involved in the biosynthesis of core LPS. By contrast in Enterobacteriaceae containing GlcN in their outer core LPS the two reactions are performed by two different enzymes.     

Molecular Basis for Galactosylation of Core Fucose Residues in Invertebrates: IDENTIFICATION OF CAENORHABDITIS ELEGANS N-GLYCAN CORE ��1,6-FUCOSIDE ��1,4-GALACTOSYLTRANSFERASE GALT-1 AS A MEMBER OF A NOVEL GLYCOSYLTRANSFERASE FAMILY*

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.     

Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

Background

The enzymatic hydrolysis of α−mannosides is catalyzed by glycoside hydrolases (GH), termed α−mannosidases. These enzymes are found in different GH sequence–based families. Considerable research has probed the role of higher eukaryotic “GH38” α−mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 α−mannosidase II, which has been shown to be a retaining α−mannosidase that targets both α−1,3 and α−1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)₅(GlcNAc)₂ hybrid N-glycans to GlcNAc(Man)₃(GlcNAc)₂. Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 α−mannosidases whose activity and specificity is unknown.

Methodology/Principal Findings

Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an α−mannosidase with specificity for α−1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 Å resolution and in complex with the inhibitor swainsonine (K _i 18 µM) at 2.6 Å, reveals a canonical GH38 five-domain structure in which the catalytic “–1” subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn²⁺ ion. In contrast, the “leaving group” subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity.

Conclusions/Significance

Although the in vivo function of this streptococcal GH38 α−mannosidase remains unknown, it is shown to be an α−mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases.     

Molecular and Biochemical Characterization of a Novel ��-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis

Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.     

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc₂) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc₂ and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc₂ as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.     

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5-7.5

The oligomerization of β-lactoglobulin (βLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of βLg variants A and B over a pH range of 2.5–7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k_off) for βLg dimer dissociation. At pH 2.5 k_off is low (0.008 and 0.009 s⁻¹), but at higher pH (6.5 and 7.5) k_off is considerably greater (>0.1 s⁻¹). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K_D^2-1 fell close to or within the protein concentration range of ∼5 to ∼45 μM, and at ∼45 μM the dimer predominated. No species larger than the dimer could be detected. The K_D^2-1 increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding βLg play a key role in the thermodynamics and kinetics of dimer association/dissociation.     

Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α1,3-fucose residues (Man₃XylFuc(GlcNAc)₂). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan₃(GlcNAc)₂ inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man₃XylFuc(GlcNAc)₂ and (GlcNAc)₂Man₃XylFuc(GlcNAc)₂ structures.     

Functions of the ��, ��, and �� Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α₂β₂γ₂ hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α₂β₂γ₂ and α₂β₂ forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the acid hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner.     

Accumulation of Free Oligosaccharides and Tissue Damage in Cytosolic α-Mannosidase (Man2c1)-deficient Mice

Free Man_7–9GlcNAc₂ is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man₅GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man_8–9GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man_8–9GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman''s capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.     

Learning/Memory Impairment and Reduced Expression of the HNK-1 Carbohydrate in ��4-Galactosyltransferase-II-deficient Mice

The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the Gal β1-4GlcNAc structure, and it is strongly expressed in the central nervous system. In behavioral tests, the β4GalT-II^-/- mice showed normal spontaneous activity in a novel environment, but impaired spatial learning/memory and motor coordination/learning. Immunohistochemistry showed that the amount of HNK-1 carbohydrate was markedly decreased in the brain of β4GalT-II^-/- mice, whereas the expression of polysialic acid was not affected. Furthermore, mice deficient in glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of the HNK-1 carbohydrate, also showed impaired spatial learning/memory as described in our previous report, although their motor coordination/learning was normal as shown in this study. Histological examination showed abnormal alignment and reduced number of Purkinje cells in the cerebellum of β4GalT-II^-/- mice. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that the glycans synthesized by β4GalT-II have essential roles in higher brain functions, including some that are HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is important for their biological activities, stability, transport, and clearance from circulation, and cell-surface glycans participate in cell-cell and cell-extracellular matrix interactions. In the central nervous system, several specific carbohydrate epitopes, including polysialic acid (PSA),³ the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides play indispensable roles in neuronal generation, cell migration, axonal outgrowth, and synaptic plasticity (1). Functional analyses of the glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc), neolactosamine core (Galβ1-3GlcNAc), and polylactosamine (Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice in β4-galactosyltransferase-I (β4GalT-I) (2, 3), β4GalT-V (4), β3-N-acetylglucosaminyl-transferase-II (β3GnT-II) (5), β3GnT-III (Core1-β3GnT) (6), β3GnT-V (7), and Core2GnT (8). However, the roles of these glycan backbone structures in the nervous system have not been examined except the olfactory sensory system (9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the β4-galactosylation of glycoproteins and glycolipids; the β4GalTs transfer galactose (Gal) from UDP-Gal to a nonreducing terminal N-acetylglucosamine (GlcNAc) of N- and O-glycans with a β-1,4-linkage. The β4GalT family has seven members (β4GalT-I to VII), of which at least five have similar Galβ1-4GlcNAc-synthesizing activities (10, 11). Each β4GalT has a tissue-specific expression pattern and substrate specificity with overlapping, suggesting each β4GalT has its own biological role as well as redundant functions. β4GalT-I and β4GalT-II share the highest identity (52% at the amino acid level) among the β4GalTs (12), suggesting these two galactosyltransferases can compensate for each other. β4GalT-I is strongly and ubiquitously expressed in various non-neural tissues, whereas β4GalT-II is strongly expressed in neural tissues (13, 14). Indeed, the β4GalT activity in the brain of β4GalT-I-deficient (β4GalT-I^-/-) mice remains as high as 65% of that of wild-type mice, and the expression levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal (15). These results suggest β4GalTs other than β4GalT-I, like β4GalT-II, are important in the nervous system.Among the β4GalT family members, only β4GalT-I^-/- mice have been examined extensively; this was done by us and another group. We reported that glycans synthesized by β4GalT-I play various roles in epithelial cell growth and differentiation, inflammatory responses, skin wound healing, and IgA nephropathy development (2, 16-18). Another group reported that glycans synthesized by β4GalT-I are involved in anterior pituitary hormone function and in fertilization (3, 19). However, no other nervous system deficits have been reported in these mice, and the role of the β4-galactosylation of glycoproteins and glycolipids in the nervous system has not been fully examined.In this study, we generated β4GalT-II^-/- mice and examined them for behavioral abnormalities and biochemical and histological changes in the central nervous system. β4GalT-II^-/- mice were impaired in spatial learning/memory and motor coordination/learning. The amount of HNK-1 carbohydrate was markedly decreased in the β4GalT-II^-/- brain, but PSA expression was not affected. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that glycans synthesized by β4GalT-II have essential roles in higher brain functions, including ones that are HNK-1 carbohydrate-dependent and ones that are independent of HNK-1.