Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays.

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein–glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

A major branch of glycobiology and glycan-focused biomedicine studies the interaction between carbohydrates and other biopolymers, most importantly, glycan-binding proteins. Today, this research into glycan-biopolymer interaction is unthinkable without glycan arrays, tools that enable high-throughput analysis of carbohydrate interaction partners. Glycan arrays offer many applications in basic biochemical research, for example, defining the specificity of glycosyltransferases and lectins such as immune receptors. Biomedical applications include the characterization and surveillance of influenza strains, identification of biomarkers for cancer and infection, and profiling of immune responses to vaccines. Here, we review major applications of glycan arrays both in basic and applied research. Given the dynamic nature of this rapidly developing field, we focus on recent findings.

Summary LSH, a protein related to the SNF2 family of chromatin-remodelling ATPases, is essential for the correct establishment of DNA methylation levels and patterns in plants and mammalian cells. However, some of the phenotypes resulting from LSH deficiency cannot be explained easily by defects in DNA methylation. Here we show that LSH-deficient mouse and human fibroblasts show reduced viability after exposure to ionizing radiation and repair DNA double-strand breaks less efficiently than wild-type cells. A more detailed characterisation of this phenotype revealed that, in the absence of LSH, the histone variant H2AX is not efficiently phosphorylated in response to DNA damage. This results in impaired recruitment of MDC1 and 53BP1 proteins to DNA double-strand breaks and compromises phosphorylation of checkpoint kinase CHK2. Furthermore, we demonstrate that the ability of LSH to hydrolyse ATP is necessary for efficient phosphorylation of H2AX at DNA double-strand breaks and successful repair of DNA damage. Taken together, our data reveal a previously unsuspected role of LSH ATPase in the maintenance of genome stability in mammalian somatic cells, which is independent of its function in de novo DNA methylation during development.

The identification of immunogenic glycotopes that render glycoconjugate vaccines protective is key to improving vaccine efficacy. Synthetic oligosaccharides are an attractive alternative to the heterogeneous preparations of purified polysaccharides that most marketed glycoconjugate vaccines are based on. To investigate the potency of semi- synthetic glycoconjugates, we chose the least efficient serotype in the current pneumococcal conjugate vaccine Prevnar 13™, Streptococcus pneumoniae serotype 3 (ST3). Glycan arrays containing synthetic ST3 repeating unit oligosaccharides were used to screen a human reference serum for antibodies and to define the recognition site of two ST3-specific protective monoclonal antibodies. The glycan array screens identified a tetrasaccharide that was selected for in-depth immunological evaluation. The tetrasaccharide-CRM197 carrier protein conjugate elicited protective immunity as evidenced by opsonophagocytosis assays and protection against pneumonia caused by ST3 in mice. Formulation of the defined protective lead candidate glycotope has to be further evaluated to elicit optimal long-term immunity.

Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Some components of the glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6-, 3,4-, or 2,3-positions. A trans-2,3-linked pyruvate is present on the CPS of the Gram-positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.

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