Kinetics Of Selectin/Ligand Interactions

Selectin/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes. Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells tethering to and rolling on the vessel wall.Each selectin contains a single N-terminal lectin (Lee) domain, followed by an epidermal growth factor (EGF)-like domain, a series of short consensus repeats (CRs), a transmembrane region (TM), and a cytoplasmic tail (Cyto). The functionality of each domain remains unclear. Not only the length and orientation of selectins play a critical role in leukocyte rolling, but does the amino acid mutation of selectins also greatly affect the efficiency of selectin mediated cell adhesion. Besides, receptor-based competitive inhibition is an appealing therapeutic strategy that holds promise for the treatment of a wide variety of diseases related to the dysfunction of cell adhesion.To examine how the molecular structures and reaction environments affect the features that favor selectin-mediated adhesions, a micropipette aspiration assay was used to measure the bindings of selectin/ligand. By using different protein coupling modes, selectins of different length and selectins mutated in specific amino acid, effects of molecular orientation, length and amino acid mutation were tested. Meanwhile, we measured the competitive inhibtions of soluble selectin to the bindings of membrane bound selectin with HL-60 cell surface ligands.Reverse kinetic rate and binding affinity constants were predicted by comparing the measured data with a small system probabilistic model. Results suggested that the molecular length, orientation and amino acid mutation had significant influences on the binding affinities but not the reverse kinetic rates of the selectin/ligand bindings, indicating that selectins must project Lee domain well above the membrane in accessible positions to mediate optimal adhesion of HL-60 cell. It was also demonstrated that acid mutation in Lec/EGF domain can drastically alter E-selectin bindings and greatly reduce the binding affinity of E-selectin to its ligands. Finally, extracellular selectin constructs were more potent than Lec/EGF in competitive inhibition selectin mediated cell adhesion. Therefore, CR domains may contribute to theenhanced binding of selectins to their ligands.The outcomes further our understanding of selectin/ligand binding and provide a basis for quantitative descriptions of interactions between flowing cells and the vessel wall under physiological conditions.