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Glycobiology

Thu, 29 Oct 2009 08:38:43 PDT

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Thu, 29 Oct 2009 08:38:43 PDT

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Thu, 29 Oct 2009 08:38:43 PDT

Metabolic glycoengineering: Sialic acid and beyond

Du, J., Meledeo, M A., Wang, Z., Khanna, H. S, Paruchuri, V. D P, Yarema, K. J — Thu, 29 Oct 2009 08:38:43 PDT

This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems – such as the human brain – through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous – and sometime quite unexpected – biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the "druggability" of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated "scaffold-dependent" activities, are summarized.

Galectin-1 stimulates monocyte chemotaxis via the p44/42 MAP kinase pathway and a pertussis toxin-sensitive pathway

Malik, R. K J, Ghurye, R. R, Lawrence-Watt, D. J, Stewart, H. J S — Thu, 29 Oct 2009 08:38:43 PDT

Galectin-1, the prototype of a family of β-galactoside-binding proteins, has been implicated in a wide variety of biological processes. Data presented herein show that galectin-1 stimulates monocyte migration in a dose-dependent manner but is not chemotactic for macrophages. Galectin-1-induced monocyte chemotaxis is blocked by lactose and inhibited by an anti-galectin-1 antibody but not by nonspecific antibodies. Furthermore, galectin-1-mediated monocyte migration was significantly inhibited by MEK inhibitors in a rapid, time-dependent manner suggesting that MAP kinase pathways are involved in galectin-1. Migration was also almost completely blocked by pertussis toxin implying G-protein involvement in the galectin-1-induced chemotaxis. These results demonstrate a role for galectin-1 in monocyte chemotaxis which differs from galectin-3 in that macrophages are nonresponsive. Furthermore, our observations suggest that galectin-1 may be involved in chemoattraction at sites of inflammation in vivo and may contribute to disease processes such as atherosclerosis.

A novel role for Gtb1p in glucose trimming of N-linked glycans

Quinn, R. P, Mahoney, S. J, Wilkinson, B. M, Thornton, D. J, Stirling, C. J — Thu, 29 Oct 2009 08:38:43 PDT

Glucosidase II (GluII) is a glycan-trimming enzyme active on nascent glycoproteins in the endoplasmic reticulum (ER). It trims the middle and innermost glucose residues (Glc2 and Glc1) from N-linked glycans. The monoglucosylated glycan produced by the first GluII trimming reaction is recognized by calnexin/calreticulin and serves as the signal for entry into this folding pathway. GluII is a heterodimer of and β subunits corresponding to yeast Gls2p and Gtb1p, respectively. While Gls2p contains the glucosyl hydrolase active site, the Gtb1p subunit has previously been shown to be essential for the Glc1 trimming event. Here we demonstrate that Gtb1p also determines the rate of Glc2 trimming. In order to further dissect these activities we mutagenized a number of conserved residues across the protein. Our data demonstrate that both the MRH and G2B domains of Gtb1p contribute to the Glc2 trimming event but that the MRH domain is essential for Glc1 trimming.

A simple micro-method for determining precise oligosaccharidic specificity of mannose-binding lectins

Debray, H., Coddeville, B., Bomfim, L. R, Ramos, M. V — Thu, 29 Oct 2009 08:38:43 PDT

A simple and inexpensive method was developed to rapidly define the specificity of mannose-specific lectins toward oligomannoside-type structures. The method involved the interaction of a mixture of N-[¹⁴C]-acetylated glycoasparagines, prepared by exhaustive pronase digestion of bovine pancreatic ribonuclease B and N-[¹⁴C]-acetylation with [¹⁴C]-acetic anhydride and containing all the possible oligomannoside-type N-glycans, with the lectin immobilized on Sepharose-4B. After exhaustive desalting, the obtained fractions were separated by high-performance thin-layer chromatography on silica gel plates and visualized by autoradiography with intensifying screen. As an example of the usefulness of this method, the fine specificity of artocarpin, the mannose-specificity lectin isolated from seeds of jackfruit (Artocarpus integrifolia) toward oligomannoside-type structures is presented. On the basis of such a determination, the best oligomannosidic ligand recognized by a mannose-specific lectin can be selected for studies of crystal structures of the lectin in complex with the defined ligand. Furthermore, some of these immobilized lectins, after definition of their precise specificities with the method, could represent valuable tools for the fractionation and characterization of oligomannose-type structures, present in complex mixtures.

Sialic acid content of tissue-specific gp96 and its potential role in modulating gp96-macrophage interactions

Thu, 29 Oct 2009 08:38:43 PDT

Cancer-derived heat shock protein gp96 induces a tumor-specific protective immune response primarily mediated by cytotoxic T lymphocytes (CTL) directed toward cancer-associated peptides associated with gp96. Both innate and adaptive immune responses have been demonstrated using a cell culture-based signaling mechanism. When used as an extraneous vaccine, one critical interaction which must occur for an immune response to be generated is the interaction between gp96 and the antigen presenting cell (APC) surface receptors (CD91, SR-A, TLR-2, and TLR-4). Our previous study concluded that gp96 purified from various rat and human prostate cancers is differentially glycosylated based on the amino and neutral monosaccharide content, and it was postulated that the monosaccharides may play a role in its biological activity. In this report, we report differences in the cancer-specific sialic acid content of gp96 purified from normal rat prostate compared to two rat prostate cancers, MAT-LyLu and Dunning G, as well as between two human prostate cancer cells, LnCaP and DU145. We also examined the modulatory effect of sialic acid residues on the binding of gp96 to APCs and its subsequent activation. Our results supported the contention that significant differences in the sialic acid content exist between Dunning G, MAT-LyLu, and normal rat prostate gp96, which affected its binding and biochemical activity to APCs. We therefore postulate that varied glycans of HPS96, a hitherto neglected structural component, may play a pivotal role in its anticancer activity. We suggest that construction of the glycan tree is a key to identification of the necessary and sufficient elements in the structure–function activity of HSP96.

Phosphoethanolamine is located at the 6-position and not at the 7-position of the distal heptose residue in the lipopolysaccharide from Neisseria meningitidis

Thu, 29 Oct 2009 08:38:43 PDT

Previous studies on LPS from Neisseria meningitidis strains M992B, the immunotype L6 strain, NMB, the type strain, a candidate LPS vaccine strain 6275z, and an extensively used clinical strain M986 had suggested that the location of the phosphoethanolamine (PEtn) residue was the 7-position of the distal heptose residue (HepII) of the inner-core oligosaccharide (OS). In all cases, this was only established by chemical methods, methylation linkage analyses. In this study, we have used standard NMR techniques to unequivocally show that the PEtn residue is actually located at the 6-position and not at the 7-position of the HepII residue in all of these strains. The 6-PEtn transferase genes were sequenced and their translated amino acid sequences were shown to be greater than 96% identical to that of the Lpt6 transferase from the L4 immunotype strain, which has been shown to transfer PEtn to the 6-position of the distal heptose residue. We discuss the implications of these findings with respect to the immunotyping scheme for the meningococci and in the context of LPS-based vaccine development.

A novel fucosyl glycosphingolipid of brine shrimp that is highly sensitive to endoglycoceramidase

Thu, 29 Oct 2009 08:38:43 PDT

Endoglycoceramidase (EGCase; EC 3.2.1.123) is a glycohydrolase that hydrolyzes the glycosidic linkage between the oligosaccharide and ceramide of various glycosphingolipids. We previously reported that hydra produced EGCase to digest glycosphingolipids of brine shrimp (Artemia salina), a type of aquatic crustacean used as a diet for the culture of hydra (Horibata Y, Sakaguchi K, Okino N, Iida H, Inagaki M, Fujisawa T, Hama Y, Ito M. 2004. J Biol Chem. 279:33379-33389). We report here that a major glycosphingolipid of brine shrimp is unique in structure and highly sensitive to EGCase. The glycosphingolipid was extracted from freshly hatched brine shrimp by Folch's partition, followed by mild alkaline hydrolysis and purification with a Sep-Pak plus silica cartridge. The structure of brine shrimp glycosphingolipid was determined by gas chromatography, gas chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, and ¹H-NMR spectrometry to be GlcNAc1-2Fuc1-3Manβ1-4Glcβ1-1'Cer. Two major molecular species of the glycosphingolipid were identified; the sugar and sphingoid base of each were the same but the major fatty acid was C22:0 and 2-hydroxy C22:0, respectively. This is the first report describing the glycosphingolipid that has an internal fucosyl residue substituted with 1-2 N-acetylglucosaminyl residue. This study also suggests the biological relevance of the glycosphingolipid as a dietary source of hydra which possesses EGCase as a digestion enzyme.

A plant class V chitinase from a cycad (Cycas revoluta): Biochemical characterization, cDNA isolation, and posttranslational modification

Thu, 29 Oct 2009 08:38:43 PDT

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)₃ from the substrate (GlcNAc)₆ through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain

Thu, 29 Oct 2009 08:38:43 PDT

Trypanosoma cruzi relies on highly galactosylated molecules as virulence factors and the enzymes involved in sugar biosynthesis are potential therapeutic targets. The synthesis of UDP-galactose in T. cruzi requires the activity of phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate. Several enzymes that participate in carbohydrate metabolism in trypanosomes are confined to specialized peroxisome-like organelles called glycosomes. The majority of glycosomal proteins contain peroxisome-targeting signals (PTS) at the COOH- or at the amino-terminus, which drive their transport to glycosomes. We had previously identified the T. cruzi PGM gene (TcPGM) and demonstrated that it encodes a functional enzyme. Here, we show that, in contrast to yeast and mammalian cells, TcPGM resides in glycosomes of the parasite. However, no classical PTS1 or PTS2 motif is present in its sequence. We investigated glycosomal targeting by generating T. cruzi cell lines expressing different domains of TcPGM fused to the green fluorescent protein (GFP). The analysis of the subcellular localization of fusion proteins revealed that an internal targeting signal of TcPGM, residing between amino acid residues 260 and 380, is capable of targeting GFP to glycosomes. These results demonstrate that, in T. cruzi, PGM import into glycosomes is mediated by a novel non-PTS domain that is located internally in the protein.

Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis

Thu, 29 Oct 2009 08:38:43 PDT

Surfactant protein D (SP-D), a lectin that recognizes carbohydrates via its C-type carbohydrate recognition domains (CRDs), regulates Mycobacterium tuberculosis (M.tb)–macrophage interactions via recognition of M.tb mannosylated cell wall components. SP-D binds to, agglutinates, and reduces phagocytosis and intracellular growth of M.tb. Species-specific variations in the CRD amino acid sequence contribute to carbohydrate recognition preferences and have been exploited to enhance the antimicrobial properties of SP-D in vitro. Here, we characterized the binding interaction between several wild-type and mutant SP-D neck + CRD trimeric subunits (NCRDs) and pathogenic and nonpathogenic mycobacterial species. Specific amino acid substitutions (i.e., the 343-amino-acid position) that flank the carbohydrate binding groove led to significant increases in binding of only virulent and attenuated M.tb strains and to a lesser extent M. marinum, whereas there was negligible binding to M. avium complex and M. smegmatis. Moreover, a nonconserved mutation at the critical 321-amino-acid position (involved in Ca²⁺ coordination) abrogated binding to M.tb and M. marinum. We further characterized the binding of NCRDs to the predominant surface-exposed mannosylated lipoglycans of the M.tb cell envelope. Results showed a binding pattern that is dependent on the nature of the side chain of the 343-amino-acid position flanking the SP-D CRD binding groove and the nature of the terminal mannosyl sugar linkages of the mycobacterial lipoglycans. We conclude that the 343 position is critical in defining the binding pattern of SP-D proteins to M.tb and its mannosylated cell envelope components.

Structural analysis of chondroitin sulfate from Scyliorhinus canicula: A useful source of this polysaccharide

Gargiulo, V., Lanzetta, R., Parrilli, M., De Castro, C. — Thu, 29 Oct 2009 08:38:44 PDT

Chondroitin sulfate (CS), a constituent of proteoglycans, is a key component of the connective tissues and it is widely used as a precautionary drug for joint diseases; for this reason, the increased demand of this polysaccharide has posed the problem to identify new and secure sources of this product. In this context, CS from the cartilage of the lesser spotted dogfish (Scyliorhinus canicula, a cartilaginous fish) was isolated and investigated through chemical and spectroscopical techniques. The structural elucidation was performed on the entire polysaccharide and confirmed analyzing the products obtained via ABC lyase treatment. As a result, its compositional analysis disclosed the occurrence of CS-A, CS-C, CS-D, and CS-0S motifs in the ratio of 41, 32, 19.8, and 8.2%, respectively. Additionally, two different glycopeptides were isolated and characterized via NMR, providing information on the linkage oligosaccharide region joining the glycosaminoglycan chain to the core protein. Therefore, chondroitin sulfate from Scyliorhinus canicula appears very similar to that isolated from shark, a cartilaginous and taxonomically related fish, with the main difference residing in the major percentage of the CS-A motif. In the light of the results obtained, Scyliorhinus canicula chondroitin sulfate possesses a chemical structure compatible for the formulation of commercial and pharmaceutical products.

Sialic acid feeding aged rats rejuvenates stimulated salivation and colon enteric neuron chemotypes

Sprenger, N., Julita, M., Donnicola, D., Jann, A. — Thu, 29 Oct 2009 08:38:44 PDT

Old age is linked to numerous changes of body functions such as salivation, gastrointestinal motility, and permeability all linked to central and enteric nervous system decline. Thus, gut motility and barrier functions suffer. Sialic acid plays a key role in the nervous system at large and for many receptor functions specifically. Decreased sialylation in the elderly suggests an endogenous sialic acid deficit. We used a rat model of aging, to ask whether sialic acid feeding would affect (i) stimulated salivation, (ii) gut functions, and (iii) sialic acid levels and neuronal markers in brain and gut. We observed reduced levels of pilocarpine-stimulated salivation in old versus young rats and restored this function by sialic acid feeding. Brain ganglioside bound sialic acid levels were found lower in aged versus young rats, and sialic acid feeding partly restored the levels. The hypothalamic expression of cholinergic and panneuronal markers was reduced in aged rats. The expression of the nitrergic marker nNOS was increased upon sialic acid feeding in aged rats. Neither fecal output nor gut permeability was different between young and aged rats studied here, and sialic acid feeding did not alter these parameters. However, the colonic expression of specific nervous system markers nNOS and Uchl1 and the key enzyme for sialic acid synthesis GNE were differentially affected in young and aged rats by sialic acid feeding indicating that regulatory mechanisms change with age. Investigation of sialic acid supplementation as a functional nutrient in the elderly may help those who suffer from disorders of reduced salivation. Further research is needed to understand the differential effects of sialic acid feeding in young and aged rats.

Caenorhabditis elegans galectins LEC-6 and LEC-1 recognize a chemically synthesized Gal{beta}1-4Fuc disaccharide unit which is present in Protostomia glycoconjugates

Thu, 29 Oct 2009 08:38:44 PDT

Galβ1-4GlcNAc is thought to be a common disaccharide unit preferentially recognized by vertebrate galectins. Eight-amino-acid residues conserved in proteins belonging to the galectin family have been suggested to be responsible for recognition. Meanwhile, we isolated and analyzed endogenous N-glycans of Caenorhabditis elegans that were captured by a C. elegans galectin LEC-6 and demonstrated that the unit of recognition for LEC-6 is a Gal-Fuc disaccharide, though the linkage between these residues was not confirmed. In the present study, we chemically synthesized Galβ1-4Fuc and Galβ1-3Fuc labeled with 2-aminopyridine (PA) and demonstrated that LEC-6 interacts with PA-Galβ1-4Fuc more strongly than PA-Galβ1-3Fuc by frontal affinity chromatography (FAC). Galβ1-4Fuc also inhibited hemagglutination caused by LEC-6 more strongly than Galβ1-3Fuc. FAC analysis using LEC-6 point mutants revealed that some of the conserved amino acid residues which have proven to be important for the recognition of Galβ1-4GlcNAc are not necessary for the binding to Galβ1-4Fuc. Another major C. elegans galectin, LEC-1, also showed preferential binding to Galβ1-4Fuc. These results suggest that Galβ1-4Fuc is the endogenous unit structure recognized by C. elegans galectins, which implies that C. elegans glycans and galectins may have co-evolved through an alteration in the structures of C. elegans glycans and a subsequent conversion in the sugar-binding mechanism of galectins. Furthermore, since glycans containing the Galβ1-4Fuc disaccharide unit have been found in organisms belonging to Protostomia, this unit might be a common glyco-epitope recognized by galectins in these organisms.

Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells

Thu, 29 Oct 2009 08:38:44 PDT

We have recently reported that the CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 induces proliferation, migration, and invasion of the Huh7 human hepatoma cells through its G-protein-coupled receptor CXCR4 and that glycosaminoglycans (GAGs) are involved in these events. Here, we demonstrate by surface plasmon resonance that the chemokine binds to GAG mimetics obtained by grafting carboxylate, sulfate or acetate groups onto a dextran backbone. We also demonstrate that chemically modified dextrans inhibit SDF-1/CXCL12-mediated in vitro chemotaxis and anchorage-independent cell growth in a dose-dependent manner. The binding of GAG mimetics to the chemokine and their effects in modulating the SDF-1/CXCL12 biological activities are mainly related to the presence of sulfate groups. Furthermore, the mRNA expression of enzymes involved in heparan sulfate biosynthesis, such as exostosin-1 and -2 or N-deacetylase N-sulfotransferases remained unchanged, but heparanase mRNA and protein expressions in Huh7 cells were decreased upon GAG mimetic treatment. Moreover, decreasing heparanase-1 mRNA levels by RNA interference significantly reduced SDF-1/CXCL12-induced extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Therefore, we suggest that GAG mimetic effects on SDF-1/CXCL12-mediated hepatoma cell chemotaxis may rely on decreased heparanase expression, which impairs SDF-1/CXCL12's signaling. Altogether, these data suggest that GAG mimetics may compete with cellular heparan sulfate chains for the binding to SDF-1/CXCL12 and may affect heparanase expression, leading to reduced SDF-1/CXCL12 mediated in vitro chemotaxis and growth of hepatoma cells.

Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa

Thu, 29 Oct 2009 08:38:44 PDT

Glycoconjugates expressed on gastric mucosa play a crucial role in host–pathogen interactions. The FUT2 enzyme catalyzes the addition of terminal (1,2)fucose residues, producing the H type 1 structure expressed on the surface of epithelial cells and in mucosal secretions of secretor individuals. Inactivating mutations in the human FUT2 gene are associated with reduced susceptibility to Helicobacter pylori infection. H. pylori infects over half the world's population and causes diverse gastric lesions, from gastritis to gastric cancer. H. pylori adhesion constitutes a crucial step in the establishment of a successful infection. The BabA adhesin binds the Le^b and H type 1 structures expressed on gastric mucins, while SabA binds to sialylated carbohydrates mediating the adherence to inflamed gastric mucosa. In this study, we have used an animal model of nonsecretors, Fut2-null mice, to characterize the glycosylation profile and evaluate the effect of the observed glycan expression modifications in the process of H. pylori adhesion. We have demonstrated expression of terminal difucosylated glycan structures in C57Bl/6 mice gastric mucosa and that Fut2-null mice showed marked alteration in gastric mucosa glycosylation, characterized by diminished expression of (1,2)fucosylated structures as indicated by lectin and antibody staining and further confirmed by mass spectrometry analysis. This altered glycosylation profile was further confirmed by the absence of Fuc(1,2)-dependent binding of calicivirus virus-like particles. Finally, using a panel of H. pylori strains, with different adhesin expression profiles, we have demonstated an impairment of BabA-dependent adhesion of H. pylori to Fut2-null mice gastric mucosa, whereas SabA-mediated binding was not affected.

Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions

Thu, 29 Oct 2009 08:38:44 PDT

The interactions of glycosaminoglycans (GAGs) with proteins underlie a wide range of important biological processes. However, the study of such binding reactions has been hampered by the lack of a simple frontline analysis technique. Previously, we have reported that cold plasma polymerization can be used to coat microtiter plate surfaces with allyl amine to which GAGs (e.g., heparin) can be noncovalently immobilized retaining their ability to interact with proteins. Here, we have assessed the capabilities of surface coats derived from different ratios of allyl amine and octadiene (100:0 to 0:100) to support the binding of diverse GAGs (e.g., chondroitin-4-sulfate, dermatan sulfate, heparin preparations, and hyaluronan) in a functionally active state. The Link module from TSG-6 was used as a probe to determine the level of functional binding because of its broad (and unique) specificity for both sulfated and nonsulfated GAGs. All of the GAGs tested could bind this domain following their immobilization, although there were clear differences in their protein-binding activities depending on the surface chemistry to which they were adsorbed. On the basis of these experiments, 100% allyl amine was chosen for the generation of a microtiter plate-based "sugar array"; X-ray photoelectron spectroscopy revealed that similar relative amounts of chondroitin-4-sulfate, dermatan sulfate, and heparin (including two selectively de-sulfated derivatives) were immobilized onto this surface. Analysis of four unrelated proteins (i.e., TSG-6, complement factor H, fibrillin-1, and versican) illustrated the utility of this array to determine the GAG-binding profile and specificity for a particular target protein.

Stability of N-glycan profiles in human plasma

Gornik, O., Wagner, J., Pucic, M., Knezevic, A., Redzic, I., Lauc, G. — Thu, 29 Oct 2009 08:38:44 PDT

Glycan heterogeneity was shown to be associated with numerous diseases and glycan analysis has a great diagnostic potential. Recently, we reported high biological variability of human plasma N-glycome at the level of population. The observed variations were larger than changes reported to be associated with some diseases; thus, it was of great importance to examine the temporal constancy of human N-glycome before glycosylation changes could be routinely analyzed in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during 5 days. N-Linked glycans, released from plasma proteins, were separated using hydrophilic interaction high-performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of N-glycome in a single individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1 were observed. The average coefficient of variation was 5.6%. These variations were comparable to those observed when analytical procedure was tested for its precision. Good stability of plasma N-glycome in healthy individuals implies that glycosylation is under significant genetic control. Changes observed in glycan profiles are consequence of environmental influences and physiologic responses and therefore have a significant diagnostic potential.

Modeling a congenital disorder of glycosylation type I in C. elegans: A genome-wide RNAi screen for N-glycosylation-dependent loci

Thu, 29 Oct 2009 08:38:44 PDT

Inefficient glycosylation caused by defective synthesis of lipid-linked oligosaccharide donor results in multi-systemic syndromes known as congenital disorders of glycosylation type I (CDG-I). Strong loss of function mutations are embryonic lethal, patients with partial losses of function are occasionally born but are very ill, presenting with defects in virtually every tissue. CDG-I clinical expression varies considerably and ranges from very mild to severe, and the underlying cause of the variable clinical features is not yet understood. We postulate that accompanying defects in an individual's genetic background enhance the severity of CDG-I clinical phenotypes. Since so many protein structures and functions are compromised in CDG-I illnesses, the gene products that are dependent on N-linked glycosylation which cause lethality or particular symptoms are difficult to resolve. The power of genetic silencing that is a characteristic of C. elegans has allowed us to systematically dissect the complex glycosylation phenotype observed in CDG-I patients into specific glycan-dependent gene products. To accomplish this, we inhibited glycosylation with a sub-phenotypic dose of tunicamycin, reduced single genes by RNA interference, and then sought loci where the combination caused a synthetic or dramatically enhanced phenotype. This screen has identified genes in C. elegans that require N-linked glycans to function properly as well as candidate gene homologues that may enhance the clinical severity of CDG-I disorders in humans.

Glycome-DB.org: A portal for querying across the digital world of carbohydrate sequences

Ranzinger, R., Frank, M., von der Lieth, C.-W., Herget, S. — Thu, 29 Oct 2009 08:38:44 PDT

Despite ongoing harmonization efforts, the major carbohydrate sequence databases following the first initiative in this field, CarbBank, are still isolated islands, with mechanisms for automatic structure exchange and comparison largely missing. This unfavorable situation has been overcome with a systematic data integration effort, resulting in the GlycomeDB, a meta-database for public carbohydrate sequences. It contains at present 35,056 unique structures in GlycoCT encoding, referencing more than 100,000 external records from 1845 different taxonomic sources. We have created a user-friendly, web-based graphical interface which allows taxonomic and structural data to be entered and searched for. The structural search possibilities include substructure search, similarity search, and maximum common substructure. A novel search refinement mechanism allows the assembly of complex queries. With GlycomeDB (www.glycome-db.org), it is now possible to use a single portal to access all digitally encoded, public structural data in glycomics and to perform complex queries with the help of a web-based user interface.

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

Thu, 29 Oct 2009 08:38:44 PDT

Author index

Thu, 29 Oct 2009 08:38:44 PDT

Glycobiology

Mon, 05 Oct 2009 21:39:40 PDT

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Mon, 05 Oct 2009 21:39:40 PDT

Meeting Announcements

Mon, 05 Oct 2009 21:39:40 PDT

Awards Announcements

Mon, 05 Oct 2009 21:39:40 PDT

Structural insights into what glycan arrays tell us about how glycan-binding proteins interact with their ligands

Taylor, M. E, Drickamer, K. — Mon, 05 Oct 2009 21:39:40 PDT

Screening of glycan arrays represents a powerful, high-throughput approach to defining oligosaccharide ligands for glycan-binding receptors, commonly referred to as lectins. Correlating results from such arrays with structural analysis of receptor–ligand complexes provide one way to validate the arrays. Using examples drawn from the family of proteins that contain C-type carbohydrate-recognition domains, this review illustrates how information from the arrays reflects the way that selectivity and affinity for glycan ligands is achieved. A range of binding profiles is observed, from very restricted binding to a small set of structurally similar ligands to binding of broad classes of ligands with related terminal sugars and even to failure to bind any of the glycans on an array. These outcomes provide insights into the importance of multiple factors in defining the selectivity of these receptors, including the presence of conformationally defined units in some oligosaccharide ligands, local and extended interactions between glycans and the surfaces of receptors, and steric factors that exclude binding of some ligands.

A mathematical model to derive N-glycan structures and cellular enzyme activities from mass spectrometric data

Mon, 05 Oct 2009 21:39:40 PDT

Effective representation and characterization of biosynthetic pathways of glycosylation can be facilitated by mathematical modeling. This paper describes the expansion of a previously developed detailed model for N-linked glycosylation with the further application of the model to analyze MALDI-TOF mass spectra of human N-glycans in terms of underlying cellular enzyme activities. The glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes. The use of a molecular mass cutoff and a network pruning method typically limits the model size to about 10,000 glycan structures. This allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. A synthetic mass spectrum from model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with experiment. The result of this process is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of 19 enzymes. In addition, a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. The method was applied to mass spectrometric data of normal human monocytes and monocytic leukemia (THP1) cells to derive glycosyltransferase activity changes underlying the differences in glycan structure between the normal and diseased cells. Model predictions could lead to a better understanding of the changes associated with disease states, identification of disease-associated biomarkers, and bioengineered glycan modifications.

QCM-D studies of human norovirus VLPs binding to glycosphingolipids in supported lipid bilayers reveal strain-specific characteristics

Rydell, G. E, Dahlin, A. B, Hook, F., Larson, G. — Mon, 05 Oct 2009 21:39:40 PDT

Susceptibility to norovirus infection has been linked to secretor status. Norovirus virus-like particles (VLPs; 0– 20 µg/mL) from the Norwalk (GI.1) and Dijon (GII.4) strains were assayed for binding to H type 1 and Lewis a pentaglycosylceramides, incorporated in laterally fluid supported lipid bilayers. Binding kinetics was monitored in real time in 40 µL stationary reaction chambers, using quartz crystal microbalance with dissipation (QCM-D) monitoring. Both strains displayed binding only to H type 1 and not to Lewis a glycosphingolipids, typical for epithelial cells of susceptible and resistant individuals, respectively. This binding specificity was confirmed by VLPs binding to the two glycosphingolipids chromatographed on TLC-plates. Experiments using bilayers with mixtures of H type 1 and Lewis a, with the total glycosphingolipid concentration constant at 10 wt%, showed that binding was only dependent on H type 1 concentrations and identical to experiments without additional Lewis a. Both strains showed a threshold concentration of H type 1 below which no binding was observable. The threshold was one order of magnitude higher for the Dijon strain (2 wt% versus 0.25 wt%) demonstrating that the interaction with a significantly larger number of glycosphingolipids was needed for the binding of the Dijon strain. The difference in threshold glycosphingolipid concentrations for the two strains suggests a lower affinity for the glycosphingolipid for the Dijon compared to the Norwalk strain. We propose that VLPs initially bind only a few glycosphingolipids but the binding is subsequently strengthened by lateral diffusion of additional glycosphingolipids moving into the interaction area.

Residual dipolar coupling investigation of a heparin tetrasaccharide confirms the limited effect of flexibility of the iduronic acid on the molecular shape of heparin

Jin, L., Hricovini, M., Deakin, J. A, Lyon, M., Uhrin, D. — Mon, 05 Oct 2009 21:39:40 PDT

The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca²⁺. Proton–proton and proton–carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of ¹C₄ and ²S_o conformers of iduronic acid that agreed well with the analysis based on proton–proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton–carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of ¹H-¹H NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans.

Dermatan 4-O-sulfotransferase 1 is pivotal in the formation of iduronic acid blocks in dermatan sulfate

Pacheco, B., Maccarana, M., Malmstrom, A. — Mon, 05 Oct 2009 21:39:41 PDT

Chondroitin/dermatan sulfate is a highly complex linear polysaccharide ubiquitously found in the extracellular matrix and at the cell surface. Several of its functions, such as binding to growth factors, are mediated by domains composed of alternating iduronic acid and 4-O-sulfated N-acetylgalactosamine residues, named 4-O-sulfated iduronic acid blocks. These domains are generated by the action of two DS-epimerases, which convert d-glucuronic acid into its epimer l-iduronic acid, in close connection with 4-O-sulfation. In this study, dermatan sulfate structure was evaluated after downregulating or increasing dermatan 4-O-sulfotransferase 1 (D4ST-1) expression. siRNA-mediated downregulation of D4ST-1 in primary human lung fibroblasts led to a drastic specific reduction of iduronic acid blocks. No change of epimerase activity was found, indicating that the influence of D4ST-1 on epimerization is not due to an altered expression level of the DS-epimerases. Analysis of the dermatan sulfate chains showed that D4ST-1 is essential for the biosynthesis of the disulfated structure iduronic acid-2-O-sulfate-N-acetylgalactosamine-4-O-sulfate, thus confirmed to be strictly connected with the iduronic acid blocks. Also the biologically important residue hexuronic acid-N-acetylgalactosamine-4,6-O-disulfate considerably decreased after D4ST-1 downregulation. In conclusion, D4ST-1 is a key enzyme and is indispensable in the formation of important functional domains in dermatan sulfate and cannot be compensated by other 4-O-sulfotransferases.

Genetic and biochemical modulation of sialic acid O-acetylation on group B Streptococcus: Phenotypic and functional impact

Weiman, S., Dahesh, S., Carlin, A. F, Varki, A., Nizet, V., Lewis, A. L — Mon, 05 Oct 2009 21:39:41 PDT

Group B Streptococcus (GBS) is an important human pathogen and a model system for studying the roles of bacterial glycosylation in host–microbe interactions. Sialic acid (Sia), expressed prominently in the GBS capsular polysaccharide (CPS), mimics mammalian cell surface Sia and can interact with host Sia-binding proteins to subvert immune clearance mechanisms. Our earlier work has shown that GBS partially O-acetylates CPS Sia residues and employs an intracellular O-acetylation/de-O-acetylation cycle to control the final level of this surface Sia modification. Here, we examine the effects of point mutations in the NeuD O-acetyltransferase and NeuA O-acetylesterase on specific glycosylation phenotypes of GBS, pinpointing an isogenic strain pair that differs dramatically in the degree of the O-acetyl modification (80% versus 5%) while still expressing comparable levels of overall sialylation. Using these strains, higher levels of O-acetylation were found to protect GBS CPS Sia against enzymatic removal by microbial sialidases and to impede engagement of human Siglec-9, but not to significantly alter the ability of GBS to restrict complement C3b deposition on its surface. Additional experiments demonstrated that pH-induced migration of the O-acetyl modification from the 7- to 9-carbon position had a substantial impact on GBS–Siglec-9 interactions, with 7-O-acetylation exhibiting the strongest interference. These studies show that both the degree and position of the GBS O-acetyl modification influence Sia-specific interactions relevant to the host–pathogen relationship. We conclude that native GBS likely expresses a phenotype of intermediate Sia O-acetylation to strike a balance between competing selective pressures present in the host environment.

First structural characterization of Burkholderia vietnamiensis lipooligosaccharide from cystic fibrosis-associated lung transplantation strains

Mon, 05 Oct 2009 21:39:41 PDT

This is the first structural elucidation of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia vietnamiensis, a clinically important member of Burkholderia cepacia complex, a group of over 10 opportunistic species that are highly problematic in cystic fibrosis. We have characterized a novel LOS structure extracted from two clonal strains of B. vietnamiensis isolated from a cystic fibrosis patient who underwent lung transplantation. Strains were selected from the pretransplantation and post-transplantation periods and endotoxin was extracted. Subsequent analysis interestingly revealed identical oligosaccharidic sequences, but variation in lipid A moieties. Further, both LOS fractions were tested for their immunostimulatory activity on human myelomonocytic U937 cells and for signaling on an HEK293 cell line stably expressing both TLR 4 and MD-2. We observed an increase in lipid A acylation and a resultant increase in biological activity in bio-reporter assays of TNF- secretion in the post-transplantation strain.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases

Soya, N., Shoemaker, G. K, Palcic, M. M, Klassen, J. S — Mon, 05 Oct 2009 21:39:41 PDT

The first comparative thermodynamic study of the human blood group glycosyltransferases, -(1->3)-N-acetylgalactosaminyltransferase (GTA) and -(1->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24°C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn²⁺, both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10⁴ M^–1 was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (~1 x 10⁴ M^–1), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn²⁺, both GTA and GTB recognize their respective trisaccharide products but with a low affinity ~10³ M^–1; the presence of UDP and Mn²⁺ has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

AftD, a novel essential arabinofuranosyltransferase from mycobacteria

Mon, 05 Oct 2009 21:39:41 PDT

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of -1,5-linked d-Araf residues with some -1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with -1,3-branching activity on linear -1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

Galectin-3 regulates peritoneal B1-cell differentiation into plasma cells

Mon, 05 Oct 2009 21:39:41 PDT

Extracellular galectin-3 participates in the control of B2 lymphocyte migration and adhesion and of their differentiation into plasma cells. Here, we analyzed the role of galectin-3 in B1-cell physiology and the balance between B1a and B1b lymphocytes in the peritoneal cavity. In galectin-3^–/– mice, the total number of B1a lymphocytes was lower, while B1b lymphocyte number was higher as compared to wild-type mice. The differentiation of B1a cells into plasma cells was associated with their abnormal adhesion and location on the mesentery. The B220 and CD43, constitutively expressed by B1 lymphocytes, were respectively up- and downregulated in galectin-3^–/– mice. Mononuclear cells were strongly adhered to the mesenteric membranes of both CD43^–/– and galectin-3^–/– mice, but in contrast to CD43^–/– mice, the accumulation of B1 cells in peritoneal membranes in galectin-3^–/– mice was accompanied by their functional differentiation into plasma cells. We have shown that in the absence of galectin-3, B1-cell differentiation into plasma cells is favored and the dynamic equilibrium of B1-cell populations in the peritoneum is maintained through a compensatory increase in B1b lymphocytes.

Program and Abstracts for the 2009 Meeting of the Society for Glycobiology

Mon, 05 Oct 2009 21:39:41 PDT

Glycobiology

Tue, 01 Sep 2009 08:29:19 PDT

Tue, 01 Sep 2009 08:29:20 PDT

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Tue, 01 Sep 2009 08:29:20 PDT

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Tue, 01 Sep 2009 08:29:20 PDT

A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction

Tue, 01 Sep 2009 08:29:20 PDT

Malignant transformation of epithelial cells is frequently associated with the alteration of glycosylation pathways. Tn is a common tumor-associated carbohydrate antigen present in 90% of human carcinomas and its expression correlates with metastatic potential and poor prognosis. Despite its relevance, the functional role of Tn in tumor biology has not been firmly established probably for the lack of appropriate experimental tools. Our aims were to produce highly reactive monoclonal antibodies against Tn making use of synthetically produced Tn and to test their usefulness for in vivo imaging as well as to define their potential functional activity in tumor cell spread. We immunized mice with Tn clustered on cationized BSA and screened the positive hybridomas with Tn-biotinylated alginate. Enzyme-linked immuno sorbent assay and immunofluorescence assays revealed that the most reactive anti-Tn IgM mAb (2154F12A4) selectively recognized Tn on the MCF7 breast cancer cell line since its binding to the cell membrane was completely abolished by preincubation with purified Tn. Importantly, QDot 800-conjugated mAb injected in MCF7-tumor bearing mice specifically bound to primary tumor lesions as well as to metastases in lymph nodes. In addition, this mAb was able to inhibit cancer cell adhesion to lymphatic endothelium suggesting a novel involvement of Tn in the lymphatic dissemination of cancer cells and hypothesizing future applications in inhibiting lymphatic metastases.

Effects of N-glycosylation on the activity and localization of GlcNAc-6-sulfotransferase 1

Desko, M. M, Gross, D. A, Kohler, J. J — Tue, 01 Sep 2009 08:29:20 PDT

N-Acetylglucosamine-6-sulfotransferase-1 (GlcNAc6ST-1) is a Golgi-resident glycoprotein that is responsible for sulfation of the l-selectin ligand on endothelial cells. Here, we report the sites at which GlcNAc6ST-1 is modified with N-linked glycans and the effects that each glycan has on enzyme activity, specificity, and localization. We determined that glycans are added at three of four potential N-linked glycosylation sites: N196, N410, and N428. The N428 glycan is required for the production of sulfated cell surface glycans: cells expressing a mutant enzyme lacking this glycan were unable to sulfate the sialyl Lewis X tetrasaccharide or a putative extended core 1 O-linked glycan. The N196 and N410 glycans differentially affect sulfation of two different substrates: cells that express an enzyme lacking the N410 glycan are able to sulfate the sialyl Lewis X substrate, but produce reduced levels of a sulfated peripheral lymph node addressin epitope and cells that express an enzyme lacking the N196 glycan are able to produce a sulfated peripheral lymph node addressin epitope, but are impaired in their ability to sulfate sialyl Lewis X. The glycans’ effects on enzyme activity may be mediated, in part, by changes in enzyme localization. While most mutants that lacked glycans localized normally within the Golgi, the N428A mutant and a mutant lacking all glycans were also found to localize ectopically. Altered trafficking of mutants may be associated with the mechanisms by which misglycosylated enzyme is degraded.

Chemical synthesis of 6(GlcNAc)- and 6(Gal)-O-sulfated SiaLeX tetrasaccharides in spacer-armed form

Pazynina, G., Sablina, M., Mayzel, M., Nasonov, V., Tuzikov, A., Bovin, N. — Tue, 01 Sep 2009 08:29:20 PDT

Practical synthesis of tetrasaccharide sulfates, 6^(GlcNAc)-O-Su-SiaLe^X-OCH₂CH₂CH₂NH₂ and 6^(Gal)-O-Su-SiaLe^X-OCH₂CH₂CH₂NH₂ (SuSO₃H), selectin ligands, and leu- kocyte trafficking agents is presented. Both sulfates were synthesized starting from the same precursor, protected SiaLe^x, by the conventional procedures of carbohydrate chemistry. The sulfated SiaLe^x derivative was modified at the spacer group to give 6^(Gal)-O-Su-SiaLe^x- OCH₂CH₂CH₂NH-COCH₂CH₂CCH, convenient for "click chemistry" mode conjugation with an azido carrier, particularly, for the synthesis of an immunogen.

The second bovine {beta}-galactoside-{alpha}2,6-sialyltransferase (ST6Gal II): genomic organization and stimulation of its in vitro expression by IL-6 in bovine mammary epithelial cells

Laporte, B., Gonzalez-Hilarion, S., Maftah, A., Petit, J.-M. — Tue, 01 Sep 2009 08:29:20 PDT

We have cloned a cDNA sequence encoding the second bovine β-galactoside-2,6-sialyltransferase whose sequence shares more than 75% of identity with hST6Gal II cDNA coding sequence. The bovine gene, located on BTA 11, spans over 50 kbp with five exons (E1–E5) containing the 1488 bp open reading frame and a 5'-untranslated exon (E0). The gene expression pattern reveals a specific tissue distribution (brain, lungs, spleen, salivary, and mammary glands) compared to ST6Gal I which is ubiquitously expressed. We identified for bovine ST6Gal II three kinds of transcripts which differ by their 5'-untranslated regions. Among them, two transcripts are brain specific whereas the third one is found in all of the tissues expressing the gene. Two pFlag-bST6Gal II vector constructions were separately transfected in COS-1 cells in order to express either membrane-bound or soluble active forms of ST6Gal II. Enzymatic assays with these two forms indicated that the enzyme used the LacdiNAc structure (GalNAcβ1,4GlcNAc) as a better acceptor substrate than the Type II (Galβ1-4GlcNAc) disaccharide. Moreover, the enzyme's efficiency is improved when the acceptor substrate is provided as a free oligosaccharide rather than as a protein-bound oligosaccharide. In order to investigate the potential role of ST6Gal II during the acute phase of inflammation, we used primary cultures of bovine mammary epithelial cells which were stimulated with pro-inflammatory cytokines. It appears that the ST6Gal II gene was upregulated in cells stimulated by IL-6. This result suggested that 2,6-sialylation mediated by this gene could contribute to organism's response to infections.

Role of N-glycosylation of the SEA module of rodent Muc3 in posttranslational processing of its carboxy-terminal domain

Tue, 01 Sep 2009 08:29:20 PDT

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A, and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two, or three N-glycans, but was partially inhibited when lacking four, five, and six N-glycans. In all, 2%, 48%, 85%, and 73% of the products from N3457A, N34567A, N134567A, and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

Free energy calculations of glycosaminoglycan-protein interactions

Gandhi, N. S, Mancera, R. L — Tue, 01 Sep 2009 08:29:20 PDT

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5–6 of PECAM-1 and a region of high affinity in domains 2–3, consistent with experimental data and ligand–protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum

Lienemann, M., Boer, H., Paananen, A., Cottaz, S., Koivula, A. — Tue, 01 Sep 2009 08:29:20 PDT

Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites –4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 µM whereas no binding of chitinbiose, -triose or 3'-sialyl-N-acetyllactosamine (Neu5Ac-3Galβ-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (K_d = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites –2 and –3 and particularly the stacking interactions by tryptophanes at subsites –3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNβ-4(GlcNAc)₄ by providing a counter charge through substitution of residue T133 at subsite –3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galβ-4(GlcNAc)₄.

Sugar-binding activity of the MRH domain in the ER {alpha}-glucosidase II {beta} subunit is important for efficient glucose trimming

Tue, 01 Sep 2009 08:29:20 PDT

Glucosidase II (GII) is a glycan-processing enzyme that trims two 1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first 1,3-linked glucose from Glc₂Man₉GlcNAc₂ (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc₁Man₉GlcNAc₂ (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic subunit (GII) and a tightly associated β subunit (GIIβ) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIβ (GIIβ-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIβ-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIβ-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIβ-MRH binds most strongly to the glycans with the 1,2-linked mannobiose structure. GII with the mutant GIIβ that lost the sugar-binding activity of GIIβ-MRH hydrolyzes p-nitrophenyl--glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIβ-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans

Yu, S.-Y., Wu, S.-W., Hsiao, H.-H., Khoo, K.-H. — Tue, 01 Sep 2009 08:29:20 PDT

Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

Glycobiology - recent issues

Glycobiology

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Meeting Announcements

Metabolic glycoengineering: Sialic acid and beyond

Galectin-1 stimulates monocyte chemotaxis via the p44/42 MAP kinase pathway and a pertussis toxin-sensitive pathway

A novel role for Gtb1p in glucose trimming of N-linked glycans

A simple micro-method for determining precise oligosaccharidic specificity of mannose-binding lectins

Sialic acid content of tissue-specific gp96 and its potential role in modulating gp96-macrophage interactions

Phosphoethanolamine is located at the 6-position and not at the 7-position of the distal heptose residue in the lipopolysaccharide from Neisseria meningitidis

A novel fucosyl glycosphingolipid of brine shrimp that is highly sensitive to endoglycoceramidase

A plant class V chitinase from a cycad (Cycas revoluta): Biochemical characterization, cDNA isolation, and posttranslational modification

Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain

Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis

Structural analysis of chondroitin sulfate from Scyliorhinus canicula: A useful source of this polysaccharide

Sialic acid feeding aged rats rejuvenates stimulated salivation and colon enteric neuron chemotypes

Caenorhabditis elegans galectins LEC-6 and LEC-1 recognize a chemically synthesized Gal{beta}1-4Fuc disaccharide unit which is present in Protostomia glycoconjugates

Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells

Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa

Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions

Stability of N-glycan profiles in human plasma

Modeling a congenital disorder of glycosylation type I in C. elegans: A genome-wide RNAi screen for N-glycosylation-dependent loci

Glycome-DB.org: A portal for querying across the digital world of carbohydrate sequences

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

Author index

Glycobiology

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Awards Announcements

Structural insights into what glycan arrays tell us about how glycan-binding proteins interact with their ligands

A mathematical model to derive N-glycan structures and cellular enzyme activities from mass spectrometric data

QCM-D studies of human norovirus VLPs binding to glycosphingolipids in supported lipid bilayers reveal strain-specific characteristics

Residual dipolar coupling investigation of a heparin tetrasaccharide confirms the limited effect of flexibility of the iduronic acid on the molecular shape of heparin

Dermatan 4-O-sulfotransferase 1 is pivotal in the formation of iduronic acid blocks in dermatan sulfate

Genetic and biochemical modulation of sialic acid O-acetylation on group B Streptococcus: Phenotypic and functional impact

First structural characterization of Burkholderia vietnamiensis lipooligosaccharide from cystic fibrosis-associated lung transplantation strains

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases

AftD, a novel essential arabinofuranosyltransferase from mycobacteria

Galectin-3 regulates peritoneal B1-cell differentiation into plasma cells

Program and Abstracts for the 2009 Meeting of the Society for Glycobiology

Glycobiology

Contents

Subscriptions

Meeting Announcements

A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction

Effects of N-glycosylation on the activity and localization of GlcNAc-6-sulfotransferase 1

Chemical synthesis of 6(GlcNAc)- and 6(Gal)-O-sulfated SiaLeX tetrasaccharides in spacer-armed form

The second bovine {beta}-galactoside-{alpha}2,6-sialyltransferase (ST6Gal II): genomic organization and stimulation of its in vitro expression by IL-6 in bovine mammary epithelial cells

Role of N-glycosylation of the SEA module of rodent Muc3 in posttranslational processing of its carboxy-terminal domain

Free energy calculations of glycosaminoglycan-protein interactions

Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum

Sugar-binding activity of the MRH domain in the ER {alpha}-glucosidase II {beta} subunit is important for efficient glucose trimming

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans