Fluorescence-based solid-phase assays to study glycan-binding protein interactions with glycoconjugates

Development of glycan microarray technologies have recently revealed many new features in the binding specificities of glycan-binding proteins (GBPs) including animal and plant lectins, antibodies, toxins, and pathogens, including viruses and bacteria. Printed glycan microarrays are very sensitive, robust, and require very small quantities of glycans and GBPs. However, glycan arrays have been limited mostly to chemoenzymatically synthesized oligosaccharides and N-glycans isolated from natural glycoproteins. O-Glycans and more complex glycoconjugates, such as glycopeptides or whole cells, are generally lacking from most types of glycan microarrays. Certain GBPs such as selectins, that have more complex binding specificity, require peptide components besides the glycan structure for high-affinity binding to the ligand. GBP binding assays on glycan microarrays will provide only partial information about the specificity and high-affinity ligands for those GBPs. Therefore, more "natural" glycoconjugate arrays are required to study more complex GBP-glycoconjugate interactions. We have utilized a simple fluorescence-based solid-phase assay on a microplate format to study GBP-glycoconjugate interactions. The method utilizes commercial streptavidin-coated microplates, where various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells, can be immobilized at a defined density. The binding of GBPs to immobilized ligands can be studied using fluorescently labeled GBPs or cells, or bound GBPs can be detected using fluorescently labeled anti-GBP antibodies. Our approach utilizing biotinylated and fixed cells in a solid-phase assay is a versatile method to study binding of GBPs to natural cell-surface glycoconjugates. Not only mammalian cells, but also microorganisms can be biotinylated and fixed, and adhesion of fluorescently labeled GBPs and antibodies to immobilized cells can be studied using standard streptavidin-coated microplates. Here, we present examples of fluorescence-based solid-phase assays to study P- and L-selectin and galectin-1 binding to immobilized glycopeptides, oligosaccharides, and cells. It should be noted that with the availability of complex glycoconjugates containing available primary amine groups, such as semisynthetic glycopeptides described here, that these could also be printed on covalent microarrays for interrogation by GBPs.