Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry

Abstract

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

“…Microarrays of adsorbed natural glycolipids and neoglycolipids obtained through conjugation of glycans to activated lipids have been used extensively (14,21,(32)(33)(34)(35). Glycoproteins and polysaccharides will bind to nitrocellulose when printed as aqueous solutions, albeit with varying immobilization efficiencies (11,14,36). Conditions for glycolipids have been optimized to liposomal formulations to induce positional flexibility resembling a cell membrane (21,37).…”

“…Although the basic principles of glycan procurement-isolation versus synthesis-remain, this task can more readily be performed by nonspecialized laboratories as the relevant glycan libraries are smaller; sometimes a few compounds might be enough, though other objectives might call for more than a hundred (14,(27)(28)(29). Library design can be guided by knowledge on naturally existing glycans, for example, those found in structure databases at http://csdb.glycoscience.ru and http://www.functionalglycomics.org or in glycomics publications.…”

“…Adsorption to the three-dimensional surface of nitrocellulose-coated glass slides was used from the very beginning for polysaccharides and glycoproteins (11). Microarrays of adsorbed natural glycolipids and neoglycolipids obtained through conjugation of glycans to activated lipids have been used extensively (14,21,(32)(33)(34)(35). Glycoproteins and polysaccharides will bind to nitrocellulose when printed as aqueous solutions, albeit with varying immobilization efficiencies (11,14,36).…”

“…Isolation from natural sources such as cultured pathogens, plant material, or animal tissue is one means to access glycans (12)(13)(14). Samples are often readily accessible, but isolation procedures and characterization of the final carbohydrates can be tedious (14).…”

Selected physiological activities and health promoting properties of cereal beta-glucans. A review

“…On the other hand, in a more recent study on the recognition of glucans by human dectin-1 (using the first sequence-defined glycome-scale microarray), no binding of barley beta-glucan and other mixed-linked glucans by dectin-1 was detected. This phenomenon was explained by the fact that dectin-1 binds solely to glucans with beta-(1,3)-linked linear gluco-oligosaccharides with degree of polymerization (DP)-10 or higher (Palma et al, 2015). In another in vitro study (Noss et al, 2013) the beta-glucans of different origin, molecular mass and structure induced strong cytokine response in human whole blood cultures.…”