Search

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host‐pathogen binding, and immune recognition, through interactions with glycan‐binding proteins. A key driving force for glycan‐protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH–π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH–π interaction between glycans and proteins by genetically incorporating electron‐rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N‐linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N‐acetylglucosamine‐containing glycans. The approach thus provides a general and effective way to manipulate glycan‐protein recognition for glycoscience applications. [ABSTRACT FROM AUTHOR]

Sialyl‐Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty‐two isomeric asymmetrical tri‐antennary N‐glycans presenting SLex‐Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high‐affinity ligands for E‐selectin. The N‐glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5′‐diphospho‐N‐trifluoracetylglucosamine (UDP‐GlcNHTFA) that can be transferred by branching N‐acetylglucosaminyltransferases to give, after base treatment, GlcNH2‐containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E‐selectin bound equally well to linear glycans and tri‐antennary N‐glycans presenting SLex‐Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri‐antennary N‐glycans. Furthermore, several H5 HAs preferentially bound to N‐glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N‐glycans presenting SLex. [ABSTRACT FROM AUTHOR]

Galacto-N-biose (GNB) derivatives were efficiently synthesized from galactose derivatives via a one-pot two-enzyme system containing two promiscuous enzymes from Bifidobacterium infantis: a galactokinase (BiGalK) and a d-galactosyl-β1-3-N-acetyl-d-hexosamine phosphorylase (BiGalHexNAcP). Mono-sialyl and di-sialyl galacto-N-biose derivatives were then prepared using a one-pot two-enzyme system containing a CMP-sialic acid synthetase and an α2-3-sialyltransferase or an α2-6-sialyltransferase.

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.

N‐Glycosylation, a main post‐translational modification of Immunoglobulin G (IgG), plays a significant role in modulating the immune functions of IgG. However, the precise function elucidation of IgG N‐glycosylation remains impeded due to the obstacles in obtaining comprehensive and well‐defined N‐glycans. Here, an easy‐to‐implement divergent approach is described to synthesize a 64‐membered IgG N‐glycan library covering all possible biantennary and bisected N‐glycans by reprogramming biosynthetic assembly lines based on the inherent branch selectivity and substrate specificity of enzymes. The unique binding specificities of 64 N‐glycans with different proteins are deciphered by glycan microarray technology. This unprecedented collection of synthetic IgG N‐glycans can serve as standards for N‐glycan structure identification in complex biological samples and the microarray data enrich N‐glycan glycomics to facilitate biomedical applications. [ABSTRACT FROM AUTHOR]