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

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 cellwall 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.Fil: Palma, Angelina S.. Imperial College London; Reino Unido. Universidade de Lisboa; PortugalFil: Liu, Yan. Imperial College London; Reino UnidoFil: Zhang, Hongtao. Imperial College London; Reino Unido. Jiangnan University; ChinaFil: Zhang, Yibing. Imperial College London; Reino UnidoFil: McCleary, Barry V.. Megazyme International Ireland; IrlandaFil: Yu, Guangli. Ocean University Of China; ChinaFil: Huang, Qilin. Huazhong Agricultural University; China. Wuhan University; ChinaFil: Guidolin, Leticia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Torosantucci, Antonella. Istituto Superiore Di Sanita; ItaliaFil: Wang, Denong. Sri International; Estados UnidosFil: Carvalho, Ana Luísa. Universidade de Lisboa; PortugalFil: Fontes, Carlos M. G. A.. Universidade de Lisboa; PortugalFil: Mulloy, Barbara. Imperial College London; Reino UnidoFil: Childs, Robert A.. Imperial College London; Reino UnidoFil: Feizi, Ten. Imperial College London; Reino UnidoFil: Chai, Wengang. Imperial College London; Reino Unid