Abstract
In this chapter, we describe the key steps of the “designer” oligosaccharide microarray approach we followed to prove the carbohydrate binding activity and define the oligosaccharide ligands for Dectin-1, an atypical C-type lectin-like signaling receptor of the mammalian innate immune system with a key role in anti-fungal immunity. The term “designer” microarray, which we introduced in the course of the Dectin-1 study refers to a microarray of oligosaccharide probes generated from ligand-bearing glycoconjugates to reveal the oligosaccharide ligands they harbor, so that these can be isolated and characterized. Oligosaccharide probes were generated from two polysaccharides, one that was bound by Dectin-1 and known to be rich in β1,3-glucose sequence and another that was not bound and was rich in β1,6-glucose sequence and served as a negative control. The approach involved: classic ELISA-type binding assays to select the polysaccharides; partial depolymerization of the polysaccharides by chemical hydrolysis; fractionation by size of the glucan oligosaccharides obtained and determination of their chain lengths by mass spectrometry; detection of Dectin-1 ligand-positive and ligand-negative oligosaccharides using the neoglycolipid (NGL) technology; methylation analysis of oligosaccharides to derive glucose linkage information, and incorporation of the newly generated glucan oligosaccharide probes into microarrays encompassing diverse mammalian-type and exogenous sequences for microarray analysis of Dectin-1.
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Acknowledgments
We gratefully acknowledge collaborations of our colleagues, past and present, in the Glycosciences Laboratory: Mark Stoll, Alex Lawson, and Colin Herbert; and we acknowledge Gordon Brown for the murine Dectin-1-Fc chimera. The Glycosciences Laboratory acknowledges with gratitude collaborators over the years with whom our microarray probes were studied. For grant support, we acknowledge the UK Medical Research Council, the UK Research Councils Basic Technology Grant (GR/S79268, “Glycoarrays”), Engineering and Physical Research Councils Translational Grant EP/G037604/1, and the NCI Alliance of Glycobiologists for Detection of Cancer and Cancer Risk (U01 CA128416). A.S.P. is a fellow of the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal).
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Palma, A.S. et al. (2012). Neoglycolipid-Based “Designer” Oligosaccharide Microarrays to Define β-Glucan Ligands for Dectin-1. In: Chevolot, Y. (eds) Carbohydrate Microarrays. Methods in Molecular Biology, vol 808. Humana Press. https://doi.org/10.1007/978-1-61779-373-8_23
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DOI: https://doi.org/10.1007/978-1-61779-373-8_23
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