Fluorescent Glycosylamides Produced by Microscale Derivatization of Free Glycans for Natural Glycan Microarrays

A novel strategy for creating naturally derived glycan microarrays has been developed. Glycosylamines are prepared from free reducing glycans and stabilized by reaction with acryloyl chloride to generate a glycosylamide in which the reducing monosaccharide has a closed-ring structure. Ozonolysis of the protected glycan yields an active aldehyde, to which a bifunctional fluorescent linker is coupled by reductive amination. The fluorescent derivatives are easily coupled through a residual primary alkylamine to generate glycan microarrays. This strategy preserves structural features of glycans required for antibody recognition and allows development of natural arrays of fluorescent glycans in which the cyclic pyranose structure of the reducing-end sugar residue is retained

Chemoenzymatic Probes for Detecting and Imaging Fucose-α(1-2)-galactose Glycan Biomarkers

The disaccharide motif fucose-α­(1-2)-galactose (Fucα­(1-2)­Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα­(1-2)­Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα­(1-2)­Gal glycans. We demonstrate that the approach is highly selective for the Fucα­(1-2)­Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease

Mining High-Complexity Motifs in Glycans: A New Language To Uncover the Fine Specificities of Lectins and Glycosidases

Knowledge of lectin and glycosidase specificities is fundamental to the study of glycobiology. The primary specificities of such molecules can be uncovered using well-established tools, but the complex details of their specificities are difficult to determine and describe. Here we present a language and algorithm for the analysis and description of glycan motifs with high complexity. The language uses human-readable notation and wildcards, modifiers, and logical operators to define motifs of nearly any complexity. By applying the syntax to the analysis of glycan-array data, we found that the lectin AAL had higher binding where fucose groups are displayed on separate branches. The lectin SNA showed gradations in binding based on the length of the extension displaying sialic acid and on characteristics of the opposing branches. A new algorithm to evaluate changes in lectin binding upon treatment with exoglycosidases identified the primary specificities and potential fine specificities of an α1–2-fucosidase and an α2–3,6,8-neuraminidase. The fucosidase had significantly lower action where sialic acid neighbors the fucose, and the neuraminidase showed statistically lower action where α1–2 fucose neighbors the sialic acid or is on the opposing branch. The complex features identified here would have been inaccessible to analysis using previous methods. The new language and algorithms promise to facilitate the precise determination and description of lectin and glycosidase specificities

Mining High-Complexity Motifs in Glycans: A New Language To Uncover the Fine Specificities of Lectins and Glycosidases

Knowledge of lectin and glycosidase specificities is fundamental to the study of glycobiology. The primary specificities of such molecules can be uncovered using well-established tools, but the complex details of their specificities are difficult to determine and describe. Here we present a language and algorithm for the analysis and description of glycan motifs with high complexity. The language uses human-readable notation and wildcards, modifiers, and logical operators to define motifs of nearly any complexity. By applying the syntax to the analysis of glycan-array data, we found that the lectin AAL had higher binding where fucose groups are displayed on separate branches. The lectin SNA showed gradations in binding based on the length of the extension displaying sialic acid and on characteristics of the opposing branches. A new algorithm to evaluate changes in lectin binding upon treatment with exoglycosidases identified the primary specificities and potential fine specificities of an α1–2-fucosidase and an α2–3,6,8-neuraminidase. The fucosidase had significantly lower action where sialic acid neighbors the fucose, and the neuraminidase showed statistically lower action where α1–2 fucose neighbors the sialic acid or is on the opposing branch. The complex features identified here would have been inaccessible to analysis using previous methods. The new language and algorithms promise to facilitate the precise determination and description of lectin and glycosidase specificities

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

<div><p>It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)₃ in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.</p></div

Effect of oseltamivir on virus entry.

<p>Viruses were adsorbed to cells in the presence or absence of oseltamivir for 1 hr, then the inoculum aspirated off, infection medium without drug was added and the plates incubated for 3 days to allow virus growth. Both virus and oseltamivir were titrated at 10-fold dilutions so the errors are ±1 log.</p

Binding of four viruses to the glycan array in the absence or presence of oseltamivir.

<p>BCM/2/92 (A) and OK/483/2008 (C) bind only NeuAcα2–6 glycans and there is no change in the presence of oseltamivir. OK/309/2006 (B) and OK/5342/2010 (D) show binding to many NeuAcα2–3 glycans but this binding is lost in the presence of oseltamivir, indicating it is NA, not HA, that binds to NeuAcα2–3.</p

Relationship between agglutination of turkey red blood cells and the highest binding signal on the Glycan Array.

<p>A. Hemagglutinating units (HAU, red) or fluorescent signal (RFU, blue) are shown per µg or per 100 ng viral protein, respectively, to approximately equalize the magnitude. B. HAU plotted against RFU with the trendline shown in black. Note that both axes are on a log scale. C. Plot of isoelectric point (pI) against binding avidity (the average of HAU and RFU).</p

Intact Reducing Glycan Promotes the Specific Immune Response to Lacto-N-neotetraose-BSA Neoglycoconjugates

The mammalian immune system responds to eukaryotic glycan antigens during infections, cancer, and autoimmune disorders, but the immunological bases for such responses are unclear. Conjugate vaccines containing bacterial polysaccharides linked to carrier proteins (neoglycoconjugates) have proven successful, but these often contain repeating epitopes and the reducing end of the glycan is less important, unlike typical glycan determinants in eukaryotes, which are shorter in length and may include the reducing end. Here, we have compared the effects of two linkage methods, one that opens the ring at the reducing end of the glycan, and one that leaves the reducing end closed, on the glycan specificity of the vaccine response in rabbits and mice. We immunized rabbits and mice with bovine serum albumin (BSA) conjugates of synthetic open- and closed-ring forms (OR versus CR) of a simple tetrasaccharide lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and tested reactivity to the immunogens and several related glycans in both OR and CR versions on glycan microarrays. We found that in rabbits the immune response to the CR conjugate was directed toward the glycan, whereas the OR conjugate elicited antibodies to the reducing end of the glycan and linker region but not specifically to the glycan itself. Unexpectedly, mice did not generate a glycan-specific response to the CR conjugate. Our findings indicate that the reducing end of the sugar is crucial for generation of a glycan-specific response to some eukaryotic vaccine epitopes, and that there are species-specific differences in the ability to make a glycan-specific response to some glycoconjugates. These findings warrant further investigation with regard to rational design of glycoconjugate vaccines

A/OK/3003/96 binds NeuAcα2–3 glycans and is able to infect CHO cells that lack NeuAcα2–6 sialylation.

<p>Virus was added to cell monolayers, then infection medium added and the infected cells incubated at 37°C for 18 hours. The cells were fixed and permeabilized for immunodetection of NP (green) accumulated in nuclei (blue DAPI) of infected cells. A control H1N1 virus that binds only NeuAcα2–6 glycans infected MDCK cells but not CHO cells.</p