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: The principles established above were used to assess the utility of the method for sequencing linear gluco-oligosaccharides containing different linkage types. Pullu-4 has two1,4-linkages and one 1,6-linkage (supplemental Table S3). The product-ion spectrum (Fig. 3A) can be used to assign the three individual linkages as well as their locations. The two A-ion sets derived from losses of 60/78/120 recorded from the two consecutive residues at the reducing side were identical to the fragmentation pattern for the 4-linked Glc (Table IB), corresponding to two 1,4-linkages. The ion set with -60/90/120 from C2 (m/z 341) (Table IB) can be used to assign the nonreducing terminal 1,6-linkage. Other α1,4/α1,6-linked gluco-oligosaccharides Pullu-7 (supplemental Fig. S3) and the isomeric trisaccharide pair Pano-3 and i-Pano-3 (supplemental Fig. S4) can also be readily identified.
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.