Figure 2

Outlier Motif Analysis of the fine carbohydrate specificity of microvirin. (Upper Panel) The fluorescence values for each glycan interaction exhibited by microvirin on the Consortium for Functional Glycomics Glycan Array #2340 at a concentration of 200 μg/mL were graphed in order of apparent intensity to determine the fluoresence threshold of positive interactions. Microvirin exhibited a clear demarcation between bound and unbound states on the array, and an unambiguous cut-off value of 2,319 relative fluorescence units (RFUs) was established. (Lower Panel) Outlier Motif Analysis was employed on the data set shown in (Upper Panel), according to the previously described method [40]. From this analysis it was determined that the presence of a Manα(1-2)-motif was necessary and sufficient for microvirin binding, while the presence of an intervening Manα1-2-linkage on the central arm of the chitobiose core structure of bound N-linked glycans reduced the affinity for microvirin for high-mannose structures. Using the refinement qualifier “Manα1,2 NO Manα1,2 on the central arm,” the summed motif scores for each glycan were plotted with respect to fluorescence intensity after detection with microvirin, which resulted in further segregation of high affinity carbohydrate motifs from the weaker affinity motifs. The dashed line represents the threshold for defining outliers, based on the distributions from all the glycans.