Publications

In this issue of Immunity, Wu et al. (2014) report that galectin-9 is required for the formation and stability of iTreg cells. Galectin-9 interacts with CD44 in association with TGF-β receptors to drive both Foxp3 and galectin-9 expression in a positive-feedforward loop involving Smad3 activation.

Schistosoma mansoni and other Schistosoma sp. are multicellular parasitic helminths (worms) that infect humans and mammals worldwide. Infection by these parasites, which results in developmental maturation and sexual differentiation of the worms over a period of 5-6 weeks, induces antibodies to glycan antigens expressed in surface and secreted glycoproteins and glycolipids. There is growing interest in defining these unusual parasite-synthesized glycan antigens and using them to understand immune responses, their roles in immunomodulation, and in using glycan antigens as potential vaccine targets. A key problem in this area, however, has been the lack of information about the enzymes involved in elaborating the complex repertoire of glycans represented by the schistosome glycome. Recent availability of the nuclear genome sequences for Schistosoma sp. has created the opportunity to define the glycogenome, which represents the specific genes and cognate enzymes that generate the glycome. Here we describe the current state of information in regard to the schistosome glycogenome and glycome and highlight the important classes of glycans and glycogenes that may be important in their generation.

UNLABELLED: GlycoPattern is Web-based bioinformatics resource to support the analysis of glycan array data for the Consortium for Functional Glycomics. This resource includes algorithms and tools to discover structural motifs, a heatmap visualization to compare multiple experiments, hierarchical clustering of Glycan Binding Proteins with respect to their binding motifs and a structural search feature on the experimental data. AVAILABILITY AND IMPLEMENTATION: GlycoPattern is freely available on the Web at http://glycopattern.emory.edu with all major browsers supported.

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MS(n) analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MS(n) are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.

While all viruses must transit the plasma membrane of mammalian cells to initiate infection, we know little about the complex processes involved in viral attachment, which commonly involve recognition of glycans by viral proteins. Glycan microarrays derived from both synthetic glycans and natural glycans isolated through shotgun glycomics approaches provide novel platforms for interrogating diverse glycans as potential viral receptors. Recent studies with influenza and rotaviruses using such glycan microarrays provide examples of their utility in exploring the challenging questions raised in efforts to define the complex mechanistic protein-glycan interactions that regulate virus attachment to host cells.

Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

The comparative dendritic cell (DC) response to glycoconjugates presented in soluble, phagocytosable, or non-phagocytosable display modalities is poorly understood. This is particularly problematic, as the probing of immobilized glycans presented on the surface of microarrays is a common screen for potential candidates for glycan-based therapeutics. However, the assumption that carbohydrate-protein interactions on a flat surface can be translatable to development of efficacious therapies, such as vaccines, which are delivered in soluble or phagocytosable particles, has not been validated. Thus, a preliminary investigation was performed in which mannose or glucose was conjugated to cationized bovine serum albumin and presented to DCs in soluble, phagocytosable, or non-phagocytosable display modalities. The functional DC response to the glycoconjugates was assessed via a high throughput assay. Dendritic cell phenotypic outcomes were placed into a multivariate, general linear model (GLM) and shown to be statistically different amongst display modalities when comparing similar surface areas. The GLM showed that glycoconjugates that were adsorbed to wells were the most pro-inflammatory while soluble conjugates were the least. DC interactions with mannose conjugates were found to be calcium dependent and could be inhibited via anti-DC-SIGN antibodies. The results of this study aim to resolve conflicts in reports from multiple laboratories showing differential DC profiles in response to similar, if not identical, ligands delivered via different modalities. Additionally, this study begins to bridge the gap between microarray binding data and functional cell responses by highlighting the phenotypes induced from adsorbed glycoconjugates as compared to those in solution or displayed on microparticles.

Recombinant antibodies are of profound clinical significance; yet, anti-carbohydrate antibodies are prone to undesirable cross-reactivity with structurally related-glycans. Here we introduce a new technology called Computational Carbohydrate Grafting (CCG), which enables a virtual library of glycans to be assessed for protein binding specificity, and employ it to define the scope and structural origin of the binding specificity of antibody JAA-F11 for glycans containing the Thomsen-Friedenreich (TF) human tumor antigen. A virtual library of the entire human glycome (GLibrary-3D) was constructed, from which 1,182 TF-containing human glycans were identified and assessed for their ability to fit into the antibody combining site. The glycans were categorized into putative binders, or non-binders, on the basis of steric clashes with the antibody surface. The analysis employed a structure of the immune complex, generated by docking the TF-disaccharide (Galβ1-3GalNAcα) into a crystal structure of the JAA-F11 antigen binding fragment, which was shown to be consistent with saturation transfer difference (STD) NMR data. The specificities predicted by CCG were fully consistent with data from experimental glycan array screening, and confirmed that the antibody is selective for the TF-antigen and certain extended core-2 type mucins. Additionally, the CCG analysis identified a limited number of related putative binding motifs, and provided a structural basis for interpreting the specificity. CCG can be utilized to facilitate clinical applications through the determination of the three-dimensional interaction of glycans with proteins, thus augmenting drug and vaccine development techniques that seek to optimize the specificity and affinity of neutralizing proteins, which target glycans associated with diseases including cancer and HIV.