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The Society for Glycobiology is a non-profit scholarly society devoted to the pursuit of knowledge of glycan structures and functions, and to the sharing of that knowledge among scientists worldwide.  For more information, please visit The Society for Glycobiology homepage.

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Glycobiology - current issue
<span class="paragraphSection"><div class="boxTitle">Abstract</div>The use of the emerging “omics” technologies for large scale population screening is promising in terms of predictive, preventive and personalized medicine. For Parkinson's disease, it is essential that an accurate diagnosis is obtained and disease progression can be monitored. Immunoglobulin G (IgG) has the ability to exert both anti-inflammatory and pro-inflammatory effects, and the <span style="font-style:italic;">N</span>-glycosylation of the fragment crystallizable portion of IgG is involved in this process. This study aimed to determine whether the IgG glycome could be a candidate biomarker for Parkinson's disease. Ninety-four community-based individuals with Parkinson's disease and a sex-, age- and ethnically-matched cohort of 102 individuals with mixed phenotypes, representative of a “normally” aged Caucasian controls, were investigated. Plasma IgG glycans were analyzed by ultra-performance liquid chromatography. Overall, seven glycan peaks and 11 derived traits had statistically significant differences (<span style="font-style:italic;">P</span> < 8.06 × 10<sup>−4</sup>) between Parkinson's disease cases and healthy controls. Out of the seven significantly different glycan peaks, four were selected by Akaike's Information Criterion to be included in the logistic regression model, with a sensitivity of 87.2% and a specificity of 92.2%. The study suggested that there may be a reduced capacity for the IgG to inhibit Fcγ-RIIIa binding, which would allow an increased ability for the IgG to cause antibody-dependent cell cytotoxicity and a possible state of low-grade inflammation in individuals with Parkinson's disease.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Expanded access to DNA sequencing now fosters ready detection of site-specific human genome alterations whose actual significance requires in-depth functional study to rule in or out disease-causing mutations. This is a particular concern for genomic sequence differences in glycosyltransferases, whose implications are often difficult to assess. A recent whole-exome sequencing study identifies (c.229 C > T) in the GalNAc-4-ST1 glycosyltransferase (<span style="font-style:italic;">CHST8</span>) as a disease-causing missense R77W mutation yielding the genodermatosis peeling skin syndrome (PSS) when homozygous. Cabral et al. (Genomics. 2012;99:202–208) cite this sequence change as reducing keratinocyte GalNAc-4-ST1 activity, thus decreasing glycosaminoglycan sulfation, as the mechanism for this blistering disorder. Such an identification could point toward potential clinical and/or prenatal diagnosis of a harmful medical condition. However, GalNAc-4-ST1 has minimal activity toward glycosaminoglycans, instead modifying terminal β1,4-linked GalNAc on N- and O-linked oligosaccharides on specific glycoproteins. We find expression, processing and catalytic activity of GalNAc-4-ST1 completely equivalent between wild type and (R77W) sulfotransferases. Moreover, keratinocytes have little or no GalNAc-4-ST1 mRNA, indicating that they do not express GalNAc-4-ST1. In addition, loss-of-function of GalNAc-4-ST1 primarily presents as reproductive system aberrations rather than skin effects. These findings, an allele frequency of 0.004357, and a 10-fold difference in prevalence of <span style="font-style:italic;">CHST8</span> (c.299 C > T, R77W) across different ethnic groups, suggest that this sequence represents a “passenger” distributed polymorphism, a simple sequence variant form of the enzyme having normal activity, rather than a “driver” disease-causing mutation that accounts for PSS. This study presents an example for guiding biomedical research initiatives, as well as medical and personal/family perspectives, regarding newly-identified genomic sequence differences.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1–6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, <span style="font-style:italic;">Oryza sativa</span> L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)<span style="font-style:italic;">n</span> (<span style="font-style:italic;">n</span> = 3–6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)<span style="font-style:italic;">n</span> (<span style="font-style:italic;">n</span> = 2–6) were almost proportional to the degree of polymerization of (GlcNAc)<span style="font-style:italic;">n</span>, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, <span style="font-style:italic;">Bryum coronatum</span>. <span style="font-style:italic;">FEBS J</span>. 278:3991–4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)<span style="font-style:italic;">n</span> binding, but is not essential for antifungal activity.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Selectins are a family of calcium-dependent, type I transmembrane, carbohydrate-binding glycoproteins. Selectins and their ligands are not only involved in physiological processes such as leukocyte homing and pathological processes such as cancer, but also play an essential role in the human implantation. L-selectin and its ligands participate in the adhesion of the blastocyst to the endometrium at the maternal–fetal interface. P-selectin and E-selectin are involved in immune recognition of maternal decidua to the embedded embryo as well as trophoblast migration within decidual spiral arterioles. Moreover, altered expression of selectins and their ligands are found to be associated with some abnormal pregnancies and infertilities. This review focuses on the current progress of research on the role of selectins and their ligands in the human implantation process.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>It was recently shown that <span style="font-style:italic;">Mycobacterium tuberculosis</span> produces cellulose which forms an integral part of its extracellular polymeric substances within a biofilm set-up. Using <span style="font-style:italic;">Mycobacterium smegmatis</span> as a proxy model organism, we demonstrate that <span style="font-style:italic;">M. smegmatis</span> biofilms treated with purified MSMEG_6752 releases the main cellulose degradation-product (cellobiose), detected by using ionic chromatography, suggesting that <span style="font-style:italic;">MSMEG_6752</span> encodes a cellulase. Its overexpression in <span style="font-style:italic;">M. smegmatis</span> prevents spontaneous biofilm formation. Moreover, the method reported here allowed detecting cellobiose when <span style="font-style:italic;">M. smegmatis</span> cultures were exposed to a subinhibitory dose of rifampicin. Overall, this study highlights the role of the MSMEG_6752 in managing cellulose production induced during biofilm formation and antibiotic stress response.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases—mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from <span style="font-style:italic;">Alicyclobacillus acidocaldarius</span> in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar <span style="font-style:italic;">Amphiura filiformis</span>, with high proportions of di- and tri-<span style="font-style:italic;">O</span>-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during <span style="font-style:italic;">A. filiformis</span> arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-<span style="font-style:italic;">O</span>-sulfated disaccharides. Conversely, treatment of <span style="font-style:italic;">A. filiformis</span> with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1–2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-<span style="font-style:italic;">O</span>-sulfotransferase 2 and dermatan-4-<span style="font-style:italic;">O</span>-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the <span style="font-style:italic;">A. filiformis</span> orthologue of bone morphogenetic factors, <span style="font-style:italic;">AfBMP2/4</span>. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-<span style="font-style:italic;">O</span>-sulfotransferase 2, with a corresponding increase in <span style="font-style:italic;">AfBMP2/4</span> during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for <span style="font-style:italic;">A. filiformis</span> arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1–62 and ∆1–116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant <span style="font-style:italic;">K</span><sub>d</sub>) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1–62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1–116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1–62) and galectin-3(Δ1–116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Glycosaminoglycans (GAGs) are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria <span style="font-style:italic;">Pasteurella multocida</span> Type F and <span style="font-style:italic;">Escherichia coli</span> K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, <span style="font-style:italic;">Chlorobium phaeobacteroides</span> DSM 266, which was very similar (~62% identical) to the open reading frames of the known bifunctional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant <span style="font-style:italic;">E. coli</span>-derived <span style="font-style:italic;">Chlorobium</span> enzyme preparations were found to possess <span style="font-style:italic;">bona fide</span> chondroitin synthase activity in vitro. This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in <span style="font-style:italic;">C. phaeobacteroides</span> is unexpected for several reasons including (a) a free-living nonpathogenic organism should not “need” an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5–3 billion years ago and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing glutathione S-transferase, a cM<strong>y</strong>c eptitope and the <strong>S</strong>tbd1<strong>C</strong>BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Glycomic analysis focused on sulfated <span style="font-style:italic;">O</span>-glycans was performed to identify novel serum carbohydrate tumor markers. Sulfated glycans were enriched by α-neuraminidase digestion of pyridylaminated glycans prepared from sera, followed by anion exchange chromatography. Sulfated <span style="font-style:italic;">O</span>-glycan profiles were constructed by two types of high performance liquid chromatography separation. Comparison of the profiles from 20 healthy controls with those of 11 gastric and 9 pancreatic cancer patients identified 14 marker candidates. The structures of these candidates were precisely analyzed using various methods including enzymatic digestion and mass spectrometry. The candidates comprised 9 core1 and 5 core2 glycans. All these candidates were monosulfated, and 11 were also mono- or difucosylated, and included various determinants such as 6-sulfo type2 lactosamine, 6-sulfo Lewis X, 6-sulfo Lewis Y, 3′-sulfo type1 lactosamine and 3′-sulfo Lewis A. Furthermore, among the core1 glycans, five candidates displayed a type1 and type2 lactosamine hybrid backbone. The levels of these candidate glycans in the sera from all 40 subjects were quantified using a selected reaction monitoring assay. These analyses revealed: (i) the levels of all candidates were elevated in sera of at least one or more patients; (ii) core1 candidates having type1–type2 hybrid backbones with 6-sulfo Lewis X, 6-sulfo type2 lactosamine or 3′-sulfo Lewis A were elevated in sera of variety of patients; and (iii) levels of the candidates varied widely among patients, suggesting analysis of multiple candidates will be an effective means of screening various cancers. To fully evaluate the clinical utility of these candidates, a further verification study is required.</span>
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Lectins are used as defense effector proteins against predators, parasites and pathogens by animal, plant and fungal innate defense systems. These proteins bind to specific glycoepitopes on the cell surfaces and thereby interfere with the proper cellular functions of the various antagonists. The exact cellular toxicity mechanism is in many cases unclear. Lectin CCL2 of the mushroom <span style="font-style:italic;">Coprinopsis cinerea</span> was previously shown to be toxic for <span style="font-style:italic;">Caenorhabditis elegans</span> and <span style="font-style:italic;">Drosophila melanogaster</span>. This toxicity is dependent on a single, high-affinity binding site for the trisaccharide GlcNAc(Fucα1,3)β1,4GlcNAc, which is a hallmark of nematode and insect <span style="font-style:italic;">N</span>-glycan cores. The carbohydrate-binding site is located at an unusual position on the protein surface when compared to other β-trefoil lectins. Here, we show that CCL2 forms a compact dimer in solution and in crystals. Substitution of two amino acid residues at the dimer interface, R18A and F133A, interfered with dimerization of CCL2 and reduced toxicity but left carbohydrate-binding unaffected. These results, together with the positioning of the two carbohydrate-binding sites on the surface of the protein dimer, suggest that crosslinking of N-glycoproteins on the surface of intestinal cells of invertebrates is a crucial step in the mechanism of CCL2-mediated toxicity. Comparisons of the number and positioning of carbohydrate-binding sites among different dimerizing fungal β-trefoil lectins revealed a considerable variability in the carbohydrate-binding patterns of these proteins, which are likely to correlate with their respective functions.</span>
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