Ph.D. Dissertation Defense by Scott Chesla
Wednesday, January 26, 2005
(Dr. Cheng Zhu, Chair)
"Two Dimensional (Solid Phase) Kinetic Analysis Of Fcgamma Receptor Three (CD16) Interaction With IgG"
Cellular adhesion research has recently focused on the small scale at the level of individual receptor-ligand bonds. This trend in research is primarily due to experimental advances which allow such individual bond force measurements. Here, one of these techniques, micromanipulation, has been extended to not only determine the bond force of individual receptor-ligand pairs, but also the intrinsic kinetic rates of the interaction. Using transmembrane (TM ) Fc gamma receptor III (CD16a-TM) and human IgG (hIgG), the dependence of adhesion probability on receptor-ligand expression densities, contract duration and contact area was quantitated. A probabilistic based theoretical formulation was developed and validated that relates the intrinsic molecular kinetic rates of the receptor–ligand interaction to the experimentally determined adhesion probability. This theoretical formulation describing individual receptor-ligand kinetics has also allowed direct evaluation of existing biophysical bond strength/kinetics paradigms at the extreme condition of single bonds. A force-displacement model was also developed to quantitate the force exerted on the RBC membrane transducer during the micropipette retraction process and found to be in agreement with previous work.
In addition to CD16a-TM, the kinetic rates of CD16a anchored via a glycosyl phosphatidylinositol (GPI) moeity (CD16a-GPI) and the two alleles of CD16b (NA1 and NA2) were determined for human, rabbit, and mouse IgG species. The binding affinity of these CD16 interactions to soluble IgG was also measured by traditional bulk chemistry approaches and compared to those measured via the micromanipulation protocol in which the IgG ligand is membrane bound in the solid phase. These data suggest that the membrane anchor itself can alter CD16 binding properties. This represents the first reported effect of the anchor on an intrinsic receptor property, its kinetic rates and binding affinity.
The hypothesis of a solution bulk chemistry solution based affinity conversion to a solid phase membrane bound affinity was then tested with little experimental support for its existence. Concurrent with this finding was the elucidation of receptor / ligand orientation effects on solid phase affinity. Essentially, the orientation of molecules on the surface affects the apparent molecular densities and therefore the on-rate and affinity of the interaction.