Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.
Bottom Line: The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans.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.
Affiliation: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; email@example.com firstname.lastname@example.org.Show MeSH
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Mentions: Both the murine and human Dectin-1 behaved similarly (Fig. 6A and supplemental Fig. S7) and showed a highly restricted binding profile to the higher oligomers DP-11 to DP-13 of the linear β1,3-linked probes isolated from curdlan (probes 75–77, supplemental Tables S7A/B). Binding was not detected to any other type of sequence nor to the other β1,3-containing oligosaccharides. Among the latter were chemically synthesized β1,3-linked oligosaccharides, DP-8 and DP-9, with one or two β1,6-linked mono-glucosyl branches (probes 152–156 and 158), or natural oligosaccharides isolated from branched polysaccharides containing β1,3 and β1,6-linkages up to DP-16, or those isolated from barley containing linear β1,3 and β1,4 linkages up to DP-16. The chain length requirement for Dectin-1 binding was corroborated by an “on-array” inhibition assay (supplemental Fig. S8).
Affiliation: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; email@example.com firstname.lastname@example.org.