Fig. 3

Binding of glycan probes by recombinant Siglec-11 and Siglec-16 from human, chimpanzee, and gorilla. Glycan binding assay was carried out as described in Materials and Methods. a Binding of synthetic glycan-polymer probes by Siglecs on solid phase. All recombinant Siglec-Fc proteins tested in the assay showed similar glycan binding preferences, in that the signal intensity of glycan probe binding was as follows: Neu5Acα2-6GalNAc > Neu5Acα2-8Neu5Ac > Neu5Acα2-6Galβ1-4Glc > Neu5Acα2-3Galβ1-4Glc. Wells coated with human TREM-1 (TREM1) or without any recombinant protein (None) showed negligible binding to these glycan probes and thus were appropriate as negative controls. Binding assays were carried out in triplicate wells for each combination of protein and probe. The experiment was repeated twice with consistent results, and a representative result is shown. Error bar represents standard error of the mean. b Binding of Siglecs to glycan microarray. Binding signal intensities were normalized by the root mean square method, and are represented by a heat map (higher color intensity = strong binding). Human Siglec-7 and Siglec-9 were included in the analysis as outgroups. Two different concentrations (20 or 40 μg/ml) of each Siglec-Fc were used in the binding analysis, and gave consistent binding patterns. Trees generated by unsupervised hierarchical clustering of Siglecs (top) and glycans (left), and the glycan structures (right) are shown beside the heat map. Siglec-11 and Siglec-16 from three species are clustered together