Quantitative measurement of selectin-ligand interactions: assays to identify a sweet pill in a library of carbohydrates.
Soluble oligosaccharides and glycoproteins can inhibit leukocyte adhesion during a range of vascular ailments including inflammation, thrombosis, and cancer metastasis. The design of such molecules in many cases is based on the structure of naturally occurring selectin ligands. In this case, synthetic selectin-ligand mimetics act as competitive inhibitors of cell adhesion. In an alternate approach, cell-permeable, small-molecule oligosaccharides have been shown to alter the metabolic pathways that lead to the biosynthesis of functional selectin-ligands. The addition of such molecules results in glycoproteins that are defective in their ability to bind selectins. Quantitative in vitro testing of the efficacy of the above inhibition strategies ideally requires the application of assays that mimic the in vivo physiological milieu in terms of the valency of selectin and selectin-ligands, the physiological fluid-flow conditions, and the use of blood cells. Assays that are performed in small volumes are preferable when the quantity of available inhibitor is scarce. Finally, the measurements must account for the rapid on- and off-rates of selectin-mediated binding interactions. This chapter addresses these issues by presenting methods to measure selectin function in enzyme-linked immunosorbent assay and flow cytometry-based static assays, cell-adhesion assays performed under shear flow in cone-plate viscometers, and Biacore surface plasmon resonance measurements of molecular-binding kinetics. Examples are presented where such methods are applied to measure the ability of simple oligosaccharides based on sialyl Lewis-X and complex molecules with the core-2 structure to block selectin function. Such methods may be extended to identify potent selectin antagonists in a library of carbohydrates.
Beauharnois
Mark E
ME
Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, NY, USA.
Neelamegham
Sriram
S
Matta
Khushi L
KL
D020650:Combinatorial Chemistry Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D008024:Ligands; D009504:Neutrophils / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D019087:Selectins / Q000032:analysis / Q000378:metabolism*; D000080506:Sialyl Lewis X Antigen; D020349:Surface Plasmon Resonance
D008024:Ligands; D009844:Oligosaccharides; D019087:Selectins; D000080506:Sialyl Lewis X Antigen