Direct Evidence That Neural Cell Adhesion Molecule (NCAM) Polysialylation Increases Intermembrane Repulsion and Abrogates Adhesion

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.Polysialic acid (PSA) 1 is a long, linear ␣2,8-linked carbohydrate composed of N-acetylneuraminic acid (Neu5Ac) residues (1). This carbohydrate is added post-translationally to the neural cell adhesion molecule (NCAM), which is responsible for a variety of functions, including axon pathfinding, synaptogenesis, and tissue formation in the central nervous system (2). The expression of the polysialylated form of NCAM (PSA-NCAM) peaks early in development and decreases with age. In some exceptions, such as the hippocampus, cells continue to express PSA-NCAM throughout the life of the organism. These regions of PSA expression are also associated with neural plasticity and the remodeling of neural connections (1, 2). Aberrant expression of PSA-NCAM is associated with tumor malignancy and metastasis, and the expression of PSA-NCAM has been detected in small cell carcinoma, neuroblastomas, and Wilm's tumor (3).Polysialic acid is thought to facilitate cell migration and plasticity by inhibiting cell adhesion to other cells and to the extracellular matrix, as a result of the large excluded volume of the polymer (4, 5). Electron microscopy images showed that PSA expression increased intercellular spacing by 10 -15 nm (4). The latter could be because of the inactivation of adhesion proteins or to the increased inter-membrane repulsion resulting from the confinement of the carbohydrate chains. Light scattering studies demonstrated that NCAM polysialylation doubles the hydrodynamic radius of NCAM. However, the latter results were based on calculations, using light scattering data and the assumption that the rod-like proteins were spherical. While this indicates the approximate size of the protein, the hydrodynamic radius does not quantify the effect of the carbohydrates on NCAM-mediated adhesion. In one proposed mechanism, for example, the increased repulsive pressure between the membranes is hypothesized to push the cells apart (6). Such a shif...

Similarities between heterophilic and homophilic cadherin adhesion

“…Some residual aggregation occurs in the absence of calcium (Fig. 4b) because of nonspecific adhesion or possibly because of residual adhesion by means of the putative EC3͞EC3 bond (10,24). However, in the absence of calcium, the majority of beads remain dispersed.…”

“…The putative EC3͞EC3 bond persisted, although it was substantially weaker (0.9 Ϯ 0.3 k B T) ( Table 1). C-CAD exhibited similar behavior (10,24). Based on the similar calcium dependence of the different cadherin bonds, the calcium dependence was assumed to apply to all interactions studied.…”

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

“…In the developing embryo and neonate, NCAM is modified by the addition of long chains of ␣2,8-polysialic acid (3,4). The presence of this highly hydrated and negatively charged carbohydrate polymer disrupts overall cell adhesion and allows cell migration (5)(6)(7). Polysialylation is catalyzed by the polysialyltransferases (polySTs), ST8SiaII and ST8SiaIV (8 -11).…”

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

“…In muscle, NCAM exists in multiple isoforms, which arise from alternative splicing of a single gene [67,68]. NCAM is also subject to several forms of post-translational modification: polysialylation of residues in the immunoglobulin domains is commonly found during development and has recently been shown to increase membrane repulsion and to enhance directional migration in specific cell types [69,70]. Conversely, O-linked glycosylation at the muscle specific domain (MSD) increases myoblast fusion [71].…”