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Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Palma AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W - Mol. Cell Proteomics (2015)

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.

View Article: PubMed Central - PubMed

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; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.

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Application of the glucome-microarray to analyses of two signaling molecules of the immune system. (A) Murine Dectin-1 and (B) human DC-SIGN analyzed as human Fc chimeras. (C) Comparison of the relative binding intensities of the three groups of proteins investigated: (i) mammalian lectin receptors of the immune system; (ii) monoclonal antibodies; and (iii) microbial CBMs. The heat map representation highlights the different glucan binding patterns revealed by the microarray analysis. The relative binding intensities were calculated as the percentage of the fluorescence signal intensity at 5 fmol given by the probe most strongly bound by each protein (normalized as 100%).
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Figure 6: Application of the glucome-microarray to analyses of two signaling molecules of the immune system. (A) Murine Dectin-1 and (B) human DC-SIGN analyzed as human Fc chimeras. (C) Comparison of the relative binding intensities of the three groups of proteins investigated: (i) mammalian lectin receptors of the immune system; (ii) monoclonal antibodies; and (iii) microbial CBMs. The heat map representation highlights the different glucan binding patterns revealed by the microarray analysis. The relative binding intensities were calculated as the percentage of the fluorescence signal intensity at 5 fmol given by the probe most strongly bound by each protein (normalized as 100%).

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).


Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Palma AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W - Mol. Cell Proteomics (2015)

Application of the glucome-microarray to analyses of two signaling molecules of the immune system. (A) Murine Dectin-1 and (B) human DC-SIGN analyzed as human Fc chimeras. (C) Comparison of the relative binding intensities of the three groups of proteins investigated: (i) mammalian lectin receptors of the immune system; (ii) monoclonal antibodies; and (iii) microbial CBMs. The heat map representation highlights the different glucan binding patterns revealed by the microarray analysis. The relative binding intensities were calculated as the percentage of the fluorescence signal intensity at 5 fmol given by the probe most strongly bound by each protein (normalized as 100%).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390274&req=5

Figure 6: Application of the glucome-microarray to analyses of two signaling molecules of the immune system. (A) Murine Dectin-1 and (B) human DC-SIGN analyzed as human Fc chimeras. (C) Comparison of the relative binding intensities of the three groups of proteins investigated: (i) mammalian lectin receptors of the immune system; (ii) monoclonal antibodies; and (iii) microbial CBMs. The heat map representation highlights the different glucan binding patterns revealed by the microarray analysis. The relative binding intensities were calculated as the percentage of the fluorescence signal intensity at 5 fmol given by the probe most strongly bound by each protein (normalized as 100%).
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).

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.

View Article: PubMed Central - PubMed

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; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.

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Related in: MedlinePlus