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Siglec-8, an immune-inhibitory sialoglycan binding lectin (S8), is expressed on the surface of eosinophils and mast cells, which are potent mediators of allergic inflammation. When S8 engages endogenous sialoglycan ligands, eosinophils undergo apoptosis and mast cell mediator release is inhibited. In the human airway, Siglec-8 ligands (S8L) are sialylated keratan sulfate chains carried on isoforms of the protein Deleted in Malignant Brain Tumors-1 (DMBT1), an immunoregulatory protein that we recently identified as the endogenous ligand for S8, DMBT1S8. We herein report that S8L is overexpressed in chronic rhinosinusitis with nasal polyposis (CRSwNP), a prevalent eosinophilic laden airway disease. Quantification and comparison of the degree to which DMBT1 carries the S8L by immunoblot analysis and lectin blot overlay, respectively, from nasal lavage showed that the S8L/DMBT1 ratio was significantly increased in CRSwNP vs. control or CRS patients. We identified the histological sites of S8L and DMBT1 expression in fresh surgically resected human nasal polyps. Histochemistry of diseased polyps and adjacent nondiseased middle turbinate (MT) tissue from CRSwNP demonstrated colocalization of S8L and DMBT1 with highest staining in submucosal glands >> epithelium > stoma. S8L expression was specifically elevated in the submucosal glands and epithelium of polyp tissue compared to MT. We hypothesize that expression of the isoform of DMBT1 carrying the Siglec-8 binding sialoglycan, DMBT1S8, is induced in polyps of CRSwNP specifically at the site of disease, is produced in the submucosal glands of polyps and secreted into the lumen of the sinonasal cavity as a host response to mitigate eosinophil-mediated inflammation.

Siglecs are transmembrane sialoglycan binding proteins, most of which are expressed on leukocyte subsets and have inhibitory motifs that translate cell surface ligation into immune suppression. In humans, Siglec-8 on eosinophils, mast cells and basophils and Siglec-9 on neutrophils, monocytes and some T-cells, mediate immune cell death, inhibition of immune mediator release and/or enhancement of anti-inflammatory mediator release. Endogenous sialoglycan ligands in tissues, mostly uncharacterized, engage siglecs on leukocytes to inhibit inflammation. Glycan array analyses demonstrated that Siglec-8, Siglec-9 and their mouse counterparts Siglec-F and Siglec-E (respectively) have distinct glycan binding specificities, with Siglec-8 more structurally restricted. Since siglecs are involved in lung inflammation, we studied Siglec-8 and Siglec-9 ligands in human lungs and airways. Siglec-8 ligands are in tracheal submucosal glands and cartilage but not airway epithelium or connective tissues, whereas Siglec-9 ligands are broadly distributed. Mouse airways do not have Siglec-8 ligands, whereas Siglec-9 ligands are on airways of both species. Extraction of human airways and lung followed by electrophoretic resolution and siglec blotting revealed Siglec-8 ligands in extracts of human trachea and cultured tracheal gland cells, but not parenchyma or cultured airway epithelial cells whereas Siglec-9 ligands were extracted from all airway and lung tissues and cells tested. Siglec-8 and Siglec-9 ligands in airways appear to be high molecular weight O-linked sialoglycoproteins. These data reveal differential glycan specificities of Siglec-8, Siglec-9 and their mouse counterparts Siglec-F and Siglec-E, and the tissue distributions and molecular characteristics of Siglec-8 and Siglec-9 sialoglycan ligands on human airways and lungs.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Sialic acids have a special place in vertebrate glycobiology, where they constitute the dominant terminal saccharides on many cell surface glycans. From early studies that identified sialoglycans as receptors for important pathogens and toxins to more recent discoveries of sialic acid-binding proteins essential for immune system (and other) functions in humans, sialic acids and sialoglycans have become cornerstones in understanding vertebrate glycobiology and pathology. During a remarkable 3-year period in the late 1950s, a newly minted postdoctoral fellow (Donald G. Comb) and his young mentor (Saul Roseman) made a surprising series of discoveries that put sialic acid research on sound chemical and biochemical footing. A detailed personal letter written by Dr. Roseman that describes this period of intense sialic acid discovery, complete with inserted figures, was given to one of us (Y.C.L.) several years later. The text and figures of this letter provide a look back at the enthusiasm, rigor and serendipity that led to their important findings through the eyes of one of the key figures in sialic acid research.

Gangliosides—sialylated glycosphingolipids—are the major glycoconjugates of nerve cells. The same four structures—GM1, GD1a, GD1b and GT1b—comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1–3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2–3- linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double- null mice were no different than St3gal2-single- null mice ; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild- type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.

The discipline of glycobiology contributes to our understanding of human health and disease through research, most of which is published in peer-reviewed scientific journals. Recently, legitimate discoveries in glycobiology have been used as marketing tools to help sell plant extracts termed "glyconutrients." The glyconutrient industry has a worldwide sales force of over half a million people and sells nearly half a billion dollars (USD) of products annually. Here we address the relationship between glyconutrients and glycobiology, and how glyconutrient claims may impact the public and our discipline.

Myelin-associated glycoprotein (MAG, Siglec-4) is a quantita-tively minor membrane component expressed preferentially onthe innermost myelin wrap, adjacent to the axon. It stabilizesmyelin–axon interactions by binding to complementary ligandsontheaxolemma.MAG,amemberoftheSiglecfamilyofsialicacid–binding lectins, binds specifically to gangliosides GD1aand GT1b, which are the major sialoglycoconjugates onmammalian axons. Mice with a disrupted Galgt1 gene lackUDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase(GM2/GD2 synthase) and fail to express complex brain gang-liosides, including GD1a and GT1b, instead expressing acomparable amount of the simpler gangliosides GM3, GD3,and O-acetyl-GD3. Galgt1-null mice produce similar amountsoftotalmyelincomparedtowild-typemice,butasthemiceage,theyexhibitaxondegenerationanddysmyelinationwithaccom-panyingmotorbehavioraldeficits.HerewereportthatGalgt1-null mice display progressive and selective loss of MAG fromthebrain.At1.5monthsofage,MAGexpressionwassimilarinGalgt1-null and wild-type mice. However, by 6 months of ageMAG was decreased ~60% and at 12 months of age ~70% inGalgt1-nullmicecomparedtowild-typelittermates.Expressionof the major myelin proteins (myelin basic protein and proteo-lipid protein) was not reduced in Galgt1-null mice compared towild type. MAG mRNAexpressionwasthesamein12-month-old Galgt1-null compared to wild-type mice, an age at whichMAG protein expression was markedly reduced. We concludethat the maintenance of MAG protein levels depends on thepresence of complex gangliosides, perhaps due to enhancedstability when MAG on myelin binds to its complementaryligands, GD1a and GT1b, on the apposing axon surface.Key words: Galgt1/N-acetylgalactosaminyltransferase/GD1a/GT1b/MAGIntroductionMyelin-associated glycoprotein (MAG) is a minor myelincomponent, making up ~1% of central nervous systemand ~0.1% of peripheral nervous system myelin proteins(Trapp, 1990). It is preferentially expressed on the inner-most, periaxonal myelin membrane (Trapp et al., 1989),where it functions in myelin–axon interactions (Schachnerand Bartsch, 2000). The axon is dependent on signalsfrom myelin, specifically MAG, for its cytoarchitecture,structure, and long-term stability (Bjartmar et al., 1999).Genetic ablation of MAG results in reduced axon caliber,reduced axon neurofilament spacing and phosphoryla-tion, and progressive axon degeneration (Fruttiger et al.,1995; Li et al., 1994; Montag et al., 1994; Yin et al.,1998). These observations led to the conclusion thatMAG is an important signaling molecule in myelin–axon interactions and is required for optimal long-termaxon stability (Schachner and Bartsch, 2000). In addition,MAG inhibits axon regeneration after injury (Li et al.,1996; McKerracher et al., 1994; Mukhopadhyay et al.,1994). Along with Nogo, oligodendrocyte myelin glyco-protein, and chondroitin sulfate proteoglycans, MAGcontributes to a central nervous system environment thatis highly inhibitory for nerve regeneration (Fournier andStrittmatter, 2001; Sandvig et al., 2004; Wang et al., 2002).MAG, expressed on myelin, binds to complementaryligands on the apposing axon surface. MAG is a memberof the Siglec family of sialic acid–binding Ig-family memberlectins (Crocker and Varki, 2001; Crocker et al., 1996).Siglecs share significant domain and sequence similarityand bind to sialic acid–bearing glycoconjugates withvarying specificities for the sialic acid linkage and penulti-mate saccharides. Gangliosides are the major sialoglyco-conjugates in the brain (Schnaar, 2000) (Figure 1). MAGbinds with high affinity and specificity to two majorbrain gangliosides, GD1a and GT1b, that are expressedprominently on axons (DeVries and Zmachinski, 1980)and that bear the MAG-binding terminal sequenceNeuAca1-3Galb1-3GalNAc (Collins et al., 1997; Yanget al., 1996). Mice engineered to lack a key enzyme in gang-lioside biosynthesis, UDP-N-acetyl-D-galactosami-ne:GM3/GD3 N-acetyl-D-galactosaminyltransferase (EC2.4.1.92), do not express the NeuAca1-3Galb1-3GalNAc

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.

As was pointed out in the editorial in the very first issue ofGlycobiology (Hart 1990), the study of the biology of carbohydrates and glycoconjugates dates back to the earliest foundations of biochemistry. What is now the Society for Glycobiology dates back to small meetings organized by the founding fathers of our field, which took place in the early 1960s (for a historical review, see Silbert at http://www. glycobiology.org/files/sfg_history.pdf ). The Society for Complex Carbohydrates was formalized in 1972, and it was agreed to have a Society meeting every year in the fall. In these early days, much of the work in the field was focused on the chemical and structural analyses of glycoconjugates, while the biological functions of glycans remained largely enigmatic. Given the limited technology of the period, the accomplishments of these early glycoscientists are remarkable! By the late 1980s, it was evident that glycoconjugates play key roles in nearly every aspect of biology, including major diseases that affect mankind. Indeed, a number of “glyco-focused” biotech companies were created leading to renewed interest in our rapidly growing field. In addition, technological advances allowed for meaningful research into the structure/functions of complex glycans in complex biological systems. The realization that glycans are critically important to nearly all aspects of biology led to coining of the term “glycobiology” (Rademacher et al. 1988). During this era, papers on glycoconjugates were published in a wide array of different types of journals, ranging from those focused on mostly chemistry to others focused mainly on biology. Thus, it was realized that glycobiology papers were either buried within thick issues of general biochemistry journals or diluted among papers in highly specialized journals focused mostly on immunology, neurobiology, virology, developmental biology and many other disciplines. In 1989, after discussions with Oxford University Press, plans to create the journal Glycobiology were finalized. An editorial board of leaders in all areas of our field was selected. It was planned that each issue of Glycobiology would contain invited mini-reviews, which would convey exciting breakthroughs in an easily readable format accessible to the general bioscience communities. Each issue would also have a “Glyco-Forum” section to highlight news, meeting announcements, and to provide a vehicle for sharing ideas not appropriate for formal research publications. An original aim of the Journal was to not only publish the best science in glycobiology, but also to keep publication times as rapid as possible. The first issue of Glycobiology was published in September of 1990 with Gerald W. Hart as Editor-in-Chief, and Anne Dell, Akira Kobata and DirkH. van den Eijnden serving as Executive Editors. Both the Editors and Board members were selected from all over the world, reflecting the international focus of the new journal. The first cover of the journal showed a Haworth Projection of a hexose enclosing an electron micrograph of the Golgi (Figure 1, reproduced on the cover of this issue), reflecting the importance of this organelle in the biosynthesis of complex glycans. In the early 1990s, it became apparent to the Board of Directors of the Society for Complex Carbohydrates that the Society’s name did not adequately reflect the new directions of the field into myriad diverse areas of biology. At the 22nd Annual Meeting (1993) in Puerto Rico, the membership nearly unanimously voted to both change the name of the Society to “Society for Glycobiology” and also to make Glycobiology the Society’s official journal (Hart 1994). By 1996, it was very clear that Glycobiology was not only the leading specialty journal in our field, but also thatGlycobiology ranked highly in terms of its relative impact among all biochemical journals, only just behind Journal of Biological Chemistry and Journal of Molecular Biology but ahead of Proteins, Biochemistry, Biochemical Journal and European Journal of Biochemistry (Hart 1996). Figure 1: Cover of inaugural issue. Glycobiology, 2015, vol. 25, no. 12, 1321–1322 doi: 10.1093/glycob/cwv090 Glyco-Forum

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc-derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.

Oligodendrocytes are central nervous system glial cells responsible for myelination of neuronal axons. During brain development oligodendrocyte progenitor cells progress through a series of morphologically and immunohistochemically distinct differentiation steps leading to mature myelin-producing oligodendrocytes. Much of this same differentiation sequence is expressed in vitro by primary oligodendrocyte progenitor cells, and by the clonal progenitor cell line CG-4. We report the use of highly specific monoclonal antibodies against GM1, GD1a, GD1b, GT1b, and GQ1b to determine major brain ganglioside expression and morphological distribution during CG-4 differentiation in vitro. Prominent anti-GD1b antibody stained defined a highly arborized intermediate stage of oligodendrocyte differentiation. In contrast, anti-GT1b antibody bound to discrete patches on the cell bodies of early progenitor cells and more mature oligodendrocytes, and to sites of progenitor arborization. The other anti-ganglioside antibodies tested did not bind above background levels. Cells with anti-GD1b antibody binding and morphology similar to those in differentiating CG-4 cells were detected in rat brain primary cell cultures enriched in oligodendrocyte precursors. The remarkably distinctive ganglioside immunoreactivity on differentiating oligodendrocytes suggests the possibility of a functional role for their surface expression.

Glycosylation is among the most complex posttranslational modifications with an extremely high level of diversity that has made it refractory to high-throughput analyses. Despite its resistance to high-throughput techniques, glycosylation is important in many critical cellular processes that necessitate a productive approach to their analysis. To facilitate studies in glycosylation, we developed a high-throughput lectin microarray for defining mammalian cell surface glycan signatures. Using the lectin microarray we established a binary analysis of cell binding and hierarchical organization of 24 mammalian cell lines. The array was also used to document changes in cell surface glycosylation during cell development and differentiation of primary murine immune system cells. To establish the biological and clinical importance of glycan signatures, the lectin microarray was applied in two systems. First, we analyzed the cell surface glycan signatures and were able to predict mannose-dependent tropism using a model pathogen. Second, we used the glycan signatures to identify novel lectin biomarkers for cancer stem-like cells in a murine model. Thus, lectin microarrays are an effective tool for analyzing diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, and cell surface biomarker identification.

Sialic acids are prominent termini of mammalian glycoconjugates and are key binding determinants for cell-cell recog-nition lectins. Binding of the sialic acid-dependent lectin, myelin-associated glycoprotein (MAG), to nerve cells is implicated in the inhibition of nerve regeneration after injury. Therefore, blocking MAG binding to nerve cell sialoglycoconjugates might enhance nerve regeneration. Previously, we reported that certain sialoglycoconjugates bearing N-acetylneuraminic acid (NeuAc) but not N-glycolylneuraminic acid (NeuGc) support MAG binding (Collins et al., 1997a). We now report highly efficient conversion of sialic acids on living neural cells from exclusively NeuAc to predominantly NeuGc using a novel synthetic metabolic precursor, N-glycolylmannosamine pentaacetate (Man-NGc-PA). When NG108-15 neuroblastoma-glioma hybrid cells, which normally express only NeuAc (and bind to MAG), were cultured in the presence of 1 mM ManNGcPA, they expressed 80-90% of their sialic acid precursor pool as NeuGc within 24 h. Within 5 days, 80% of their ganglioside-associated sialic acids and 70% of their glycoprotein-associated sialic acids were converted to NeuGc. Consistent with this result, treatment of NG108-15 cells with ManNGcPA resulted in nearly complete abrogation of MAG binding. These results demonstrate that ManNGcPA treatment efficiently alters the sialic acid structures on living cells, with a commensurate change in recognition by a physiologically important lectin.