Examining Galectin Binding Specificity Using Glycan Microarrays.

Glycan binding proteins (GBPs) possess the unique ability to regulate a wide variety of biological processes through interactions with highly modifiable cell surface glycans. While many studies demonstrate the impact of glycan modification on GBP recognition and activity, the relative contribution of subtle changes in glycan structure on GBP binding can be difficult to define. To overcome limitations in the analysis of GBP-glycan interactions, recent studies utilized glycan microarray platforms containing hundreds of structurally defined glycans. These studies not only provided important information regarding GBP-glycan interactions in general but have also resulted in significant insight into binding specificity and biological activity of the galectin family. We will describe the methods used when employing glycan microarray platforms to examine galectin-glycan binding specificity and function.

Synthetic Glycan Microarrays.

Structurally diverse glycans are expressed by all animate beings and exert diverse biological functions through specific interactions with glycan binding proteins (GBPs). In humans, glycan-GBP interactions are implicated in many disease-relevant processes in development, infection and immune response to bacterial and viral pathogens. Recent progress in chemical synthesis, including automated glycan assembly, has facilitated access to complex glycans that cannot be isolated from biological material. Glycan immobilization on microarrays allows rapid, multiplexed glycan-GBP interaction studies to reveal biological functions. Synthetic glycan microarrays have enabled, for instance, the identification of glycan ligands for lectins, the definition of vaccine antigens, revealed viral glycan receptors and can serve as diagnostic tools for human disease. Here, we describe the methods to fabricate custom glycan microarrays that are used to examine glycan-GBP binding specificities. Conjugation-ready synthetic glycans are covalently attached to microarray surfaces through nucleophilic linker moieties. Microarrays are incubated with GBPs, and binding events are quantitatively detected by fluorescent signals. These methods are readily adaptable to a multitude of purposes from basic research to biomedical applications.