Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy.
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.
Gestwicki
Jason E
JE
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Cairo
Christopher W
CW
Mann
David A
DA
Owen
Robert M
RM
Kiessling
Laura L
LL
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
D000588:Amines; D002264:Carboxylic Acids; D003911:Dextrans; D008024:Ligands; D019087:Selectins; C014392:carboxymethyl dextran