Mechanism of the neural cell adhesion molecule and mode of polysialic acid adhesive inhibition

The neural cell adhesion molecule (NCAM) is a cell-surface protein that binds to identical molecules on adjacent neural cells and mediates cell-cell adhesion and signal transduction. These proteins are involved in the formation of tissue during development, and in the plasticity of mature tissue. Despite their importance in neurobiology, there is no consensus with regards to the interactions responsible for NCAM binding. This thesis examines the mechanism of homophilic adhesion between opposing monolayers of the complete extracellular segments of NCAM, as well as the effects of post-translational addition of polysialic acid on adhesion.; We begin by using the Surface Force Apparatus to measure the interactions between the tandem arranged, homologous domains, that form the extracellular region of NCAM. We determine that, rather than binding in a single unique orientation, NCAM adheres in two distinct antiparallel alignments. The strongest adhesion is measured when the opposing extracellular fragments are completely interdigitated. A second adhesive complex is observed when only domains one and two of the proteins interdigitate. When the adhesive complex fails, the NCAM proteins unbind. In addition, evidence of a flexible hinge region within the protein is also found.; A characterization of the structure of membrane supported NCAM monolayers used in the surface force measurements is presented in the latter part of the thesis. Using both X-ray and neutron reflectivity, the density profiles of NCAM and polysialylated NCAM layers and their supporting lipids normal to the air-water interface are determined. These density profiles are used to estimate the dimensions of the NCAM, polysialic acid, and lipid monolayers. The profiles combined with results gathered using fluorescence correlation spectroscopy, clearly show that polysialic acid extends above the lipid layer surface and possesses a large ionic strength sensitive hydrodynamic radius, which would interfere with NCAM homophilic interactions.; Lastly, direct force measurements are presented on polysialylated NCAM proteins to determine the effects of polysialic acid on both NCAM and C-cadherin mediated adhesion. Results indicate that polysialic acid inhibits both NCAM and cadherin adhesion by increasing the intermembrane repulsion. This inhibitory effect was dependant on both polysialic acid surface density and on ionic strength.