期刊
MOLECULAR & CELLULAR PROTEOMICS
卷 14, 期 4, 页码 974-988出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M115.048272
关键词
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资金
- Wellcome Trust [WT093378MA, WT099197MA]
- UK Research Councils' Basic Technology Initiative Glycoarrays [GRS/79268]
- EPSRC [EP/G037604/1]
- NSFC-Shandong Joint Fund for Marine Science Research Centers [U1406402]
- Fundacao para a Ciencia e Tecnologia (FCT) [PTDC/QUI-QUI/112537/2009, PEst-C/EQB/LA0006/2013]
- China Scholarship Council [CSC: 2008679005]
- Fundação para a Ciência e a Tecnologia [PTDC/QUI-QUI/112537/2009] Funding Source: FCT
- EPSRC [EP/G037604/1] Funding Source: UKRI
- MRC [MC_U117533887] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [EP/G037604/1] Funding Source: researchfish
- Medical Research Council [MC_U117533887] Funding Source: researchfish
- The Francis Crick Institute [10029] Funding Source: researchfish
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 alpha- or beta-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.
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