title: Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy.
abstract: Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Cairo
authors.forename: David A
authors.initials: DA
authors.lastname: Mann
authors.forename: Robert M
authors.initials: RM
authors.lastname: Owen
authors.forename: Laura L
authors.initials: LL
authors.lastname: Kiessling
mesh_terms: D000588:Amines / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D003911:Dextrans / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008856:Microscopy, Fluorescence; D019087:Selectins / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D000588:Amines; D002264:Carboxylic Acids; D003911:Dextrans; D008024:Ligands; D019087:Selectins; C014392:carboxymethyl dextran