Abstract
Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space.
Highlights
Polysialic acid (PSA)1 is a long, linear ␣2,8-linked carbohydrate composed of N-acetylneuraminic acid (Neu5Ac) residues (1)
For PSA to block neural cell adhesion molecule (NCAM) adhesion by increasing the nonspecific repulsive force between cells, the force balance requires that the magnitude of the additional repulsion exceed the NCAM attraction at both membrane separations at which NCAM binds
These measurements show quantitatively that the post-translational modification of NCAM substantially increases the magnitude of the repulsive pressure between membranes, sufficient to abrogate both NCAM- and cadherin-mediated adhesion
Summary
Materials—1,2-Di-tridecanoyl-sn-glycero-3-phosphocholine and 1,2di-palmitoyl-sn-glycero-3-phosphoethanolamine were purchased from Avanti Polar Lipids (Alabaster, AL). 6-[9-[2,3-Bis(dodecyloxy)propyl(3,6,9-trioxanonyl-1-oxycarboxylamino)-2-[di(carboxymethyl)-amino]hexanoic acid (NTA-TRIG-DLGE) was custom synthesized by Northern Lipids, Inc. (British Columbia, Canada). 1-Octadecanethiol, HEPES, sodium nitrate, nickel sulfate, and other high purity, inorganic salts were purchased from Aldrich. Quantification of Protein Surface Densities—To determine the surface density of proteins immobilized on the NTA-TRIG-DLGE supported bilayers, we measured the amount of bound 125I-labeled protein, according to procedures described previously (22, 23). To quantify the surface densities of immobilized NCAM, PSANCAM and cadherin prepared from solutions of two proteins at molar ratios other than 1:1, we quantified the fluorescence intensities on the supported membranes. Fluorescence intensity images were taken using a ϫ100 UPLANFI oil objective (Olympus, Tokyo, Japan), on a BX60 optical microscope from Olympus (Hamburg, Germany), which was interfaced to a digital camera (Diagnostics Instruments, Sterling, MI) For these measurements, a supported bilayer displaying a mixed protein monolayer, prepared as described above, was placed in a fluid cell and mounted on the microscope stage. A solution of 1 ϫ 10Ϫ8 M fluorescein in 50 mM Tris was used both for calibration and as a standard for determining the size of the beam waist
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