Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry

PALMA AS; LIU Y; ZHANG H; ZHANG Y; MCCLEARY BV; YU G; HUANG Q; GUIDOLIN LS; CIOCCHINI AE; TOROSANTUCCI A; WANG D; CARVALHO AL; FONTES CM; MULLOY B; CHILDS RA; FEIZI T; CHAI W

MOLECULAR & CELLULAR PROTEOMICS

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2015 vol. 14 p. 974 - 988

1535-9476

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.