Denong Wang

Enhanced immune recognition of cryptic glycan markers in human tumors.

Abnormal glycosylation is one of the hallmarks of the cancer cell and is associated with tumor invasion and metastasis. The development of tumor-associated carbohydrate antigen (TACA) vaccines has been problematic due to poor immunogenicity. However, when appropriate targets can be identified, passive immunization with monoclonal antibodies (mAbs) directed against TACAs has been shown to have antitumor activity. Fas ligand (FasL) is a transmembrane protein that induces apoptosis in cells expressing its receptor, Fas. When grafted into mice, FasL-expressing tumor cells break immunologic tolerance to self-antigens and induce antibody-mediated tumor immunity. Here, five IgM mAbs were produced from mice vaccinated with FasL-expressing B16F10 mouse melanoma cells. They recognize various syngeneic and allogeneic murine tumor cell lines. One mAb, TM10, recognizes a range of human tumor cell lines, including melanoma, prostate, and ovarian cancer. It does not bind to untransformed cells. The epitopes recognized by all the mAbs were carbohydrates expressed on proteins. Using carbohydrate microarrays, the antigenic targets of TM10 were found to be high-mannose core structures of N-linked glycans. In normal cells, high-mannose clusters are hidden by extensive saccharide branching but they become exposed in cancer cells as a result of abnormal glycosylation pathways. Vaccination with FasL-expressing tumors therefore enables the immune system to break tolerance to self-antigens, allowing identification of novel TACAs that can form the basis of future humoral anticancer therapy.

Carbohydrate cluster microarrays fabricated on three-dimensional dendrimeric platforms for functional glycomics exploration.

We reported here a novel, ready-to-use bioarray platform and methodology for construction of sensitive carbohydrate cluster microarrays. This technology utilizes a three-dimensional (3-D) poly(amidoamine) starburst dendrimer monolayer assembled on glass surface, which is functionalized with terminal aminooxy and hydrazide groups for site-specific coupling of carbohydrates. A wide range of saccharides, including monosaccharides, oligosaccharides and polysaccharides of diverse structures, are applicable for the 3-D bioarray platform without prior chemical derivatization. The process of carbohydrate coupling is effectively accelerated by microwave radiation energy. The carbohydrate concentration required for microarray fabrication is substantially reduced using this technology. Importantly, this bioarray platform presents sugar chains in defined orientation and cluster configurations. It is, thus, uniquely useful for exploration of the structural and conformational diversities of glyco-epitope and their functional properties.

Chemoenzymatic synthesis and lectin array characterization of a class of N-glycan clusters.

N-Glycans are major components of many glycoproteins. These sugar moieties are frequently involved in important physiological and disease processes via their interactions with a variety of glycan-binding proteins (GBP). Clustering effect is an important feature in many glycan-lectin interactions. We describe in this paper a chemoenzymatic synthesis of novel N-glycan clusters using a tandem endoglycosidase-catalyzed transglycosylation. It was found that the internal beta-1,2-linked GlcNAc moieties in the N-glycan core, once exposed in the nonreducing terminus, was able to serve as acceptors for transglycosylation catalyzed by Endo-A and EndoM-N175A. This efficient chemoenzymatic method allows a quick extension of the sugar chains to form a class of glycan clusters in which sugar residues are all connected by native glycosidic linkages found in natural N-glycans. In addition, a discriminative enzymatic reaction at the two GlcNAc residues could be fulfilled to afford novel hybrid clusters. Lectin microarray studies revealed unusual properties in glyco-epitope expression by this panel of structurally well-defined synthetic N-glycans. These new compounds are likely valuable for functional glycomics studies to unveil new functions of both glycans and carbohydrate-binding proteins.

Photons to illuminate the universe of sugar diversity through bioarrays

In this mini-review, we summarize the photochemical approaches for developing high-throughput carbohydrate microarray technologies. Newly established methods for photo-immobilizing unmodified monosaccharides, oligosaccharides and polysaccharides onto photoactive surfaces and coupling of photoactive carbohydrates onto polymer surfaces are reviewed.

Uncovering Cryptic Glycan Markers in Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE)

Preclinical Research

Carbohydrate Microarrays as Essential Tools of Postgenomic Medicine

Publisher Summary Carbohydrates are essential biological molecules carrying important biological information. Carbohydrates are prominently displayed on the surface of cell membranes and expressed by virtually all secretory proteins in bodily fluids. This is achieved by the events of posttranslational protein modification called glycosylation. Importantly, expression of cellular glycans, in the form of either glycoproteins or glycolipids, is differentially regulated. Cell display of precise complex carbohydrates is characteristically associated with the stages or steps of embryonic development, cell differentiation, as well as transformation of normal cells to abnormally differentiated tumor or cancer cells. Sugar moieties are also abundantly expressed on the outer surfaces of the majority of viral, bacterial, protozoan, and fungal pathogens. Many sugar structures are pathogen specific, which makes them important molecular targets for pathogen recognition, diagnosis of infectious diseases, and vaccine development. Exploring the biological information content in carbohydrates is one of the current focuses of postgenomic research and technology development. Biophysical, biochemical, and immunological methods have proven very valuable in studying carbohydrate–carbohydrate and carbohydrate–protein interactions. Many well established immunochemical methods have been applied to determine the specificity and cross-reactivity of carbohydrate–antibody and carbohydrate–lectin interactions. A pressing need is, thus, the establishment of high-throughput technologies to enable the large-scale, multiplex analysis of carbohydrates and their cellular receptors. These include especially the characterization of immunological properties of carbohydrates that are important for medical applications of carbohydrate antigens and interactions of carbohydrates with other biomolecules or intact cells that play key roles in establishing comprehensive biological functions of essentially all existing living organisms.

Photo-Generation of Carbohydrate Microarrays

The unparalleled structural diversity of carbohydrates among biological molecules has been recognized for decades. Recent studies have highlighted carbohydrate signaling roles in many important biological processes, such as fertilization, embryonic development, cell differentiation and cellȁ4cell communication, blood coagulation, inflammation, chemotaxis, as well as host recognition and immune responses to microbial pathogens. In this chapter, we summarize recent progress in the establishment of carbohydrate-based microarrays and the application of these technologies in exploring the biological information content in carbohydrates. A newly established photochemical platform of carbohydrate microarrays serves as a model for a focused discussion.