An activated L-selectin mutant with conserved equilibrium binding properties but enhanced ligand recognition under shear flow

Selectins mediate the initial tethering and rolling of leukocytes on vessel walls. Adhesion by selectins is a function of both ligand recognition at e quilibrium and mechanical properties of the selectin-ligand bond under appl ied force. We describe an EGF domain mutant of L-selectin with profoundly a ugmented adhesiveness over that of native L-selectin but conserved ligand s pecificity. This mutant, termed LPL, was derived by a substitution of the E GF-like domain of L selectin with the homologous domain from P-selectin. Th e mutant bound soluble carbohydrate L-selectin ligand with affinity compara ble with that of native L-selectin but interacted with all surface-bound li gands much more readily than native L-selectin, in particular under elevate d shear flow. Tethers mediated by both native and mutant L-selectin exhibit ed similar lifetimes under a range of shear stresses, but the rate of bond formation by the mutant was at least 10-fold higher than that of native L-s electin toward distinct L-selectin ligands. Enhanced rate of bond formation by the mutant was associated with profoundly stronger rolling interactions and reduced dependence of rolling on a threshold of shear stress. This is the first demonstration that the EGF domain can modulate the binding of the lectin domain of a selectin to surface-immobilized ligands under shear flo w without affecting the equilibrium properties of the selectin toward solub le ligands.