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; firstname.lastname@example.org email@example.com.Show MeSH
Related in: MedlinePlus
Mentions: Here, we describe a strategy using the designer approach combined with negative-ion ESI-CID-MS/MS for constructing a microarray of sequence-defined gluco-oligosaccharides representing major sequences in glucans (glucome microarray) as a tool for screening glucan-recognizing proteins and assigning their recognition motifs (Fig. 1). We selected a comprehensive panel of glucan polysaccharides isolated from plants, fungi, and bacteria with different sequences to represent the glucome. We used finely tuned chemical and enzymatic methods to partially depolymerize the polysaccharides and prepare gluco-oligosaccharide fragments with different chain lengths (up to DP-13 or DP-16). We developed a ESI-CID-MS/MS method that enables linkage and sequence determination of linear or branched gluco-oligosaccharides at high-sensitivity and applied this to the sequencing of oligosaccharide fragments prepared. These sequence-defined gluco-oligosaccharides were then converted into NGL probes and used for construction of the microarray. The oligosaccharides encompassed linear sequences with homo (single) linkages: 1,2-, 1,3-, 1,4-, or 1,6- with α or β configurations; and hetero (multiple) linkages: 1,3-, 1,4, or 1,6-; also branched oligosaccharide sequences with 1,3 and 1,6-linkages.
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; firstname.lastname@example.org email@example.com.