Binding Kinetics And Microstructural Differences Of CD44 Binding To Selectins/Hyaluron

Transmembrane protein CD44 is widely expressed on a variety of cell types.The interplay between CD44 and its ligand plays a pivotal role in many pathophysiological processes including lymphocyte homing,stem cell differentiation,inflammatory cascade,tumor metastasis and others.CD44-selectins interactions are crucial for mediating cellular adhesions in classical inflammatory cascade,but the differences in binding CD44 to three different selectins remain unclear,especially for their binding kinetics and microstructural features.In addition,CD44 binding to another key ligand hyaluronic acid or HA is also key to mediate the recruitment of leukocytes in hepatic sinusoids in liver immune response.High(HHA)and low(LHA)molecular weight HAs coexist in vivo and have different immune functions but it is unknown whether these different immune effects of HHA and LHA are related to the interaction ability of CD44-HA pair.Based on these cues,this dissertation aimed to quantify the kinetic differences and microstructural bases of CD44 binding to three selectin members by integrating experimental measurements and simulation predictions,and to elucidate the interacting strength and mechanical regulation of CD44-HA system with distinct HA molecular weights.The main conclusions are as follows:1.Kinetic difference and microstructural bases of CD44-selectin interactionsBinding of CD44 to three different selectins presents significantly different knietics.Under shear flow,CD44-E-selectin interaction mediates the firm adhesion of microbeads,CD44-P-selectin interaction induces the majority of rolling adhesion and the minority of firm adhesion,and CD44-L-selectin interaction tends to inititate the transient adhesion.At molecular level,the rupture force of CD44-E-selectin binding is significantly higher than those of CD44-P-selectin and CD44-L-selectin bindings,and there is no significant difference between the latter two systems.Based on the docking and molecular dynamics simulations,it is further predicted that CD44 binds to the hinge region between E-selectin lectin and EGF domains,and the resulted complex presents the lowest binding free energy,the highest rupture force and the longest bond lifetime.While the binding sites of CD44 locate at the sides of P-selectin Lectin domain and L-selectin EGF domain,respectively,the binding free energy,rupture force and lifetime of these two systems are comparable.Molecular simulations are consistent with the experimental measurements.2.Kinetics and mechanical regulation of CD44 binding to HA with different molecular weightsSignificant differences are found in binding CD44 to HA with high(HHA)and low molecular weights(LHA).At cellular level,CD44-HA binding mediates cell adhesion more effectively under low shear stress.CD44-HHA interactions yield the stronger ability to mediate cell adhesion,whereas the adhered cells mediated by CD44-LHA interactions present the higher shear resistance.At molecular level,the rupture force of CD44-HHA complex is higher than that of CD44-LHA interaction.Moreover,CD44-HHA interaction mediates more spreading of adhesive cells,and promotes the higher expression of CD18 and CD44 as well as the more polarized distribution of CD44 under low shear stress.In conclusion,CD44-E-selectin interaction is stronger than those of P-selectin and L-selectin for CD44-selectins systems.For CD44-HAs systems,HHA presents stronger interaction to CD44,promoting the higher expressions of CD18 and CD44 and the more polarization of CD44 distribution under low shear stress.This work provides a basic insight for understanding the biological function of CD44-ligands interactions from the perspective of molecular level and structure-function relationship.