Abstract
Background:
E-selectin interactions with glycoprotein ligands mediate the initial capturing of cells out of flow.
Results:
Adopting a Biacore-based immunoprecipitation binding assay unraveled differential binding kinetics of monomeric (m)
versus
dimeric (d) E-selectin to endogenous ligands.
Conclusion:
Although mE-selectin binds transiently, dE-selectin binds with remarkably slow on- and off-rates.
Significance:
Transitioning from monomeric to dimeric E-selectin could enable fast but firm capturing of cells out of flow.
Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both
O
- and
N
-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.