Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity.

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

Novel Reversible Fluorescent Glycan Linker for Functional Glycomics.

To aid in generating complex and diverse natural glycan libraries for functional glycomics, more efficient and reliable methods are needed to derivatize glycans. Here we present our development of a reversible, cleavable bifunctional linker 3-(methoxyamino)propylamine (MAPA). As the fluorenylmethyloxycarbonate (Fmoc) version (F-MAPA), it is highly fluorescent and efficiently derivatizes free reducing glycans to generate closed-ring derivatives that preserve the structural integrity of glycans. A library of glycans were derivatized and used to generate a covalent glycan microarray using N-hydroxysuccinimide derivatization. The array was successfully interrogated by a variety of lectins and antibodies, demonstrating the importance of closed-ring chemistry. The glycan derivatization was also performed at large scale using milligram quantities of glycans and excess F-MAPA, and the reaction system was successfully recycled up to five times, without an apparent decrease in conjugation efficiency. The MAPA-glycan is also easy to link to protein to generate neoglycoproteins with equivalent glycan densities. Importantly, the MAPA linker can be reversibly cleaved to regenerate free reducing glycans for detailed structural analysis (catch-and-release), often critical for functional studies of undefined glycans from natural sources. The high conjugation efficiency, bright fluorescence, and reversible cleavage of the linker enable access to natural glycans for functional glycomics.

Desialylation in physiological and pathological processes: New target for diagnostic and therapeutic development.

Desialylation is a pivotal part of sialic acid metabolism, which initiates the catabolism of glycans by removing the terminal sialic acid residues on glycans, thereby modulating the structure and functions of glycans, glycoproteins, or glycolipids. The functions of sialic acids have been well recognized, whereas the function of desialylation process is underappreciated or largely ignored. However, accumulating evidence demonstrates that desialylation plays an important role in a variety of physiological and pathological processes. This chapter summarizes the current knowledge pertaining to desialylation in a variety of physiological and pathological processes, with a focus on the underlying molecular mechanisms. The potential of targeting desialylation process for diagnostic and therapeutic development is also discussed.

Unique Binding Specificities of Proteins toward Isomeric Asparagine-Linked Glycans.

The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine (Asn)-linked N-glycans, we synthesized a library of isomeric multiantennary N-glycans that vary in terminal non-reducing sialic acid, galactose, and N-acetylglucosamine residues, as well as core fucose. We identified specific recognition of N-glycans by several plant lectins, human galectins, influenza viruses, and Siglecs, and explored the influence of sialic acid linkages and branching of the N-glycans. These results show the unique recognition of complex-type N-glycans by a wide variety of glycan-binding proteins and their abilities to distinguish isomeric structures, which provides new insights into the biological roles of these proteins and the uses of lectins in biological applications to identify glycans.

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins.

Glycans and glycan binding proteins (GBPs or lectins) are essential components in almost every aspect of immunology. Investigations of the interactions between glycans and GBPs have greatly advanced our understanding of the molecular basis of these fundamental immunological processes. In order to better study the glycan-GBP interactions, microscope glass slide-based glycan microarrays were conceived and proved to be an incredibly useful and successful tool. A variety of methods have been developed to better present the glycans so that they mimic natural presentations. Breakthroughs in chemical biology approaches have also made available glycans with sophisticated structures that were considered practically impossible just a few decade ago. Glycan microarrays provide a wealth of valuable information in immunological studies. They allow for discovery of detailed glycan binding preferences or novel binding epitopes of known endogenous immune receptors, which can potentially lead to the discovery of natural ligands that carry the glycans. Glycan microarrays also serve as a platform to discover new GBPs that are vital to the process of infection and invasion by microorganisms. This review summarizes the construction strategies and the immunological applications of glycan microarrays, particularly focused on those with the most comprehensive sets of glycan structures. We also review new methods and technologies that have evolved. We believe that glycan microarrays will continue to benefit the growing research community with various interests in the field of immunology.

Facile and Stereo-Selective Synthesis of UDP-α-D-xylose and UDP-ß-L-arabinose Using UDP-Sugar Pyrophosphorylase.

A novel synthesis of nucleotide sugars was conducted to prepare UDP-α-D-xylose and UDP-ß-L-arabinose without utilizing protection strategies or advanced purification techniques. Sugar-1-phosphates of D-xylose and L-arabinose were synthesized from their ß-glycosylsulfonylhydrazides and evaluated as substrates for recombinant UDP-sugar pyrophosphorylases from Arabidopsis thaliana or Bifidobacterium infantis to furnish the biologically active nucleotide. The facile, three-step procedure takes advantage of substrate diversity available through chemical synthesis followed by the selectivity of enzyme catalysis. This approach increases the substrate scope of enzymatic preparation and expands access to stereopure nucleotide sugars on preparative scale. Increased production of both sugars has implications for glycoengineering and glycan production using glycosyltransferases.

Decoding glycan protein interactions by a new class of asymmetric N-glycans.

N-Glycans are normally involved in crucial physiological and disease processes by interactions with glycan-binding proteins. So far structurally defined N-glycans have been good candidates for glycan binding study. Herein, a class of homogeneous asymmetric N-glycans was synthesized by coupling glycan-oxazoline and N-glycans using EndoM N175Q catalyzed quick glycan extension. Branch-biased binding and spacial inhibition caused by the bulky group on the other branch of N-glycan were observed in glycan protein interactions involving lectins and these glycans by glycan microarray study. These new compounds are valuable for functional glycomic studies to better understand new functions of glycans and glycan-binding proteins.

L-Rhamnose Enhances the Immunogenicity of Melanoma-Associated Antigen A3 for Stimulating Antitumor Immune Responses.

Vaccines based on melanoma-associated antigens (MAGEs) present a promising strategy for tumor immunotherapy, albeit with weak immunogenicity. In this study, the xenoantigen L-rhamnose (Rha) was chemically conjugated with truncated MAGE-A3 (tMAGE-A3) to generate Rha-tMAGE-A3. The product showed good antigenicity with anti-Rha antibodies purified from human serum. FITC-labeled Rha-tMAGE-A3 was detected in THP-1 human macrophage cells via the anti-Rha antibody-dependent antigen uptake process. Furthermore, peripheral blood mononuclear cells (PBMCs) stimulated with Rha-tMAGE-A3 in the presence of anti-Rha antibodies showed better cytotoxicity toward A375 human melanoma cells surfaced by MAGE-A3 antigen compared to PBMCs stimulated with tMAGE-A3. All data reveal that linking of Rha epitopes to MAGE enhances the immunogenicity of MAGE by harnessing the immune effector functions of human naturally existing anti-Rha antibodies. Rha epitopes could become immunogenicity enhancers of tumor associated antigens in the development of tumor immunotherapies.

A General Chemoenzymatic Strategy for the Synthesis of Glycosphingolipids.

A concise, prototypical, and stereoselective strategy for the synthesis of therapeutically and immunologically significant glycosphingolipids has been developed. This strategy provides a universal platform for glycosphingolipid synthesis by block coupling of enzymatically prepared free oligosaccharideglycans to lipids using glycosyl N-phenyltrifluoroacetimidates as efficient activated intermediates. As demonstrated here, two different types of glycosphingolipids were obtained in excellent yields using the method.

Facile Enzymatic Synthesis of Ketoses.

Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.

Facile chemoenzymatic synthesis of biotinylated heparosan hexasaccharide.

A biotinylated heparosan hexasaccharide was synthesized using a one-pot multi-enzyme strategy, in situ activation and transfer of N-trifluoroacetylglucosamine (GlcNTFA) to a heparin backbone significantly improved the synthetic efficiency. The biotinylated hexasaccharide could serve as a flexible core to diversify its conversion into heparan sulfate isoforms with potential biological applications and therapeutics.

Chemical synthesis of the outer core oligosaccharide of Escherichia coli R3 and immunological evaluation.

Lipopolysaccharides (LPS), major virulence determinants in Gram-negative bacteria, are responsible for many pathophysiological responses and can elicit strong immune responses. In order to better understand the role of LPS in host-pathogen interactions and elucidate the immunogenic properties of LPS outer core oligosaccharides, an all α-linked Escherichia coli R3 outer core pentasaccharide was first synthesized with a propyl amino linker at the reducing end. This oligosaccharide was also covalently conjugated to a carrier protein (CRM197) via the reducing end propyl amino linker. Immunological analysis demonstrated that this glycoconjugate can elicit specific anti-pentasaccharide antibodies with in vitro bactericidal activity. These findings will contribute to the further exploration of this pentasaccharide antigen as a vaccine candidate.

One-pot four-enzyme synthesis of ketoses with fructose 1,6-bisphosphate aldolases from Staphylococcus carnosus and rabbit muscle.

By the action of D-fructose 1,6-bisphosphate aldolases (FruA) from rabbit muscle and Staphylococcus carnosus, various ketoses were synthesized from glyceraldehydes or other aliphatic aldehydes as acceptors in a one-pot four-enzyme system.