Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.endoglycosidase | Fc glycosylation | glycoengineered antibodies | homogeneous antibodies | sugar oxazoline

We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.

We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAVspecific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.influenza A virus | glycosylation | vaccine design I nfluenza A viruses (IAV) belong to the Orthomyxoviridae family and can circulate widely and cross interspecies barriers through the highly antigenic drift and shift of the 18 subtypes of HA and 11 subtypes of neuraminidase (NA) (1, 2). In addition, the posttranslational modification of the IAV surface proteins is important to circumvent host defense and support the virus life cycle (3).HA is a major surface glycoprotein of IAV and is involved in viral infection via binding to sialic acid (SA)-containing glycans on the surface of host cell (3). The other major glycoprotein of IAV, NA, is involved in the cleavage of SA on the host cell receptor to facilitate the release of viral particles to infect other cells (4). M2, the third surface protein of IAV, has ion channel activity to regulate virus penetration and uncoating (1). All surface proteins interact with M1 protein for virus assembly and release (5).The modification of HA and NA by N-glycosylation is important in the IAV life cycle (1, 6-8). Previously, we have shown that the glycosylation of HA affects its receptor binding, immune response, and structural stability, and glycosite 27 is essential for retaining the structural integrity of HA and its receptor binding (6, 9). In addition, using the monoglycosylated HA as immunogen, it showed a broader protection against various IAV subtypes compared with the fully glycosylated version (10). However, it is not clear whether the other specific glycosites and their glycan structures on HA regulate its functions. With regard to NA, the functional roles of its glycosylation are not well understood, though N-glycosylation was reported to stabilize the protein from protease digestion and may affect the enzyme activity (11,12). The aim of this study was to understand the functional effects of glycosylation on HA, NA, and M2, and how glycosylation of the surface proteins affects the life cycle of IAV. ResultsGlycosylation of HA at N-142. Because several glycosylation sites (glycosites 27, 40, 176, 303, and 497) on HA are ...

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120, thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

Numerous biomolecules possess α-D-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted β-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.