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: Hydrolysis of grifolan and lentinan was carried out with 0.02 m TFA at 100 °C for 12 h. After cooling to room temperature, the hydrolysate mixture was centrifuged. The supernatant was coevaporated with MeOH and fractionated by a short (1.6 × 36 cm) column of Bio-Gel P6. The high (>DP-20) and low (< DP-20) oligomer fractions, as determined by MALDI-MS, were kept separately. The precipitate and high oligomer fractions were combined and the TFA treatment procedure was repeated three times to increase the yield of low oligomers. The low oligomer fractions were combined and subjected to a final gel filtration to obtain, from grifolan, fractions 2 to 16 with Glc3 to Glc16 as major components (Grifo-3 to Grifo-16) and from lentinan fractions 2 to 13, with Glc2 to Glc13 as major components (Lenti-2 to Lenti-13). Microarray binding data with the α1,4-glucose specific TmCBM41 (Fig. 5B and supplemental Table S7B) indicated the presence of a minor α1,4-linked glucose contaminant in the lentinan fractions, particularly those containing oligomers with >DP-8 as major components. This was corroborated by MS/MS (footnote in supplemental Table S7A) and NMR (not shown).
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.