Effect Of Tyrosine Replacement And Cell Surface Microtopology And Stiffness On Kinetics Of Selectin/Ligand Interaction

Selectin/ligand interactions have been found to mediate the tethering and rolling of circulating leukocytes on vessel wall during inflammatory response. Kinetic rate, binding affinity constants, and mechanical strength of selectin/ligand bonds are critical determinants of cell rolling. These physical, chemical determinants are related to the structure of the molecules and the environment of the carrier surface. The carrier surface influences not only selectin/ligand but also other adhesion molecules, playing an essential role in cell adhesion, organization and tissue formation.The first part of this thesis delineated the structure-function relationship of a selectin ligand at the level of amino acid. All the members of the selectin family, P-, E-, and L-selectin, bind ubiquitously to PSGL-1 (P-selectin glycoprotein ligand-1). Preliminary studies found that two structures of PSGL-1 are essential for the binding to P- and L-selectin: tyrosine sulfate at residue 46, 48, and 51, and a core-2 O-glycan capped with sLex (sialyl Lewis x) at Thr-57. Modification of tyrosine replacement affected distinct reverse kinetic rates and mechanical properties of selectin/PSGL-1 bonds.To further characterize the role of the amino acids in selectin/PSGL-1 binding, a micropipette aspiration assay was used to measure the intrinsic reverse kinetic rates and affinity of selectin/mutated PSGL-1 bonds. Wild type human PSGL-1, four mutated PSGL-1 with double tyrosine substitutions at 46, 48 and 51, respectively, core-2 O-glycan, and sLex were expressed onto Chinese Hamster Ovary (CHO) cell lines by transfection. Anti selectin monoclonal antibodies were coated onto the human red blood cells using a CrCl3 protocol, which then captured selectin. The dependence of binding frequency on contact duration was measured. By fitting the measured data using an established small system probabilistic model, both kinetic rates and binding affinities were derived. The reverse kinetic rates did not vary much after tyrosine replacement. Surprisingly, the binding affinity for wild type PSGL-1 was ~7.8-16 folds as high as those for the mutants, indicating that the forward rate of the mutants were significantly decreased because of tyrosine replacement. This provides a new insight into understandings of selectin/ligand binding at amino acid level.Selectin/ligand and other adhesion molecules are expressed on the surface of carriers. To describe the influence of microtopology and stiffness of the molecular carrier on two-dimensional interaction, the second part of this thesis established a series of methods. Rosette assay was adopted to model the two-dimensional interaction of various carriers and produce bond surface. Different roughness and stiffness were observed by Scanning Electron Microscopy. To further compare the influence of these differences on adhesion, a mBFP (modified Biomembrane Force Probe) assay was established. The protein coating procedures of BFP were simplified and the multiple working procedures were developed. By fitting the measured data with the probabilistic model, kinetic rates, binding affinity and force range of 1~1000 pN could be measured. Excellent specificity were obtained. These improvements further the applications of BFP in the field of cell adhesion.