Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase.

Abstract

A phage endoneuraminidase that specifically cleaves alpha-2, 8-1inked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.The neural cell adhesion molecule (NCAM) t is a cell-surface glycoprotein that serves as a ligand in the formation of cellcell bonds (see references 9, 26, and 27 for review). The binding appears to involve the direct interaction between NCAMs on each cell, and therefore represents an example of homophilic adhesion (31). NCAM is expressed on a variety of cell types in the vertebrate embryo, including primitive neuroepithelia (36), neurons (29, 32), glial cells (14, 23), and muscle cells (15). Adhesion of neurons to muscle, glia, and other neurons has been found to be mediated at least in part by NCAM, and appears to be an essential event in the formation of nerve-muscle contacts that lead to synapses ~ Abbreviations used in this paper: endo-N, the soluble endoneuraminidase from K1F bacteriophage; NCAM, neural cell adhesion molecule.(30), guidance of axon growth cones along marginal pathways of the central nervous system (36), and the formation of neurite fascicles (29). In addition, antibodies to NCAM have been shown to alter the histogenesis of the retina in vitro (3).The complex glycan chains associated with NCAM have a very unusual structure including one or more relatively long unbranched homopolymers of alpha-2, 8-1inked sialic acid residues (6,7,12,43). Removal of the sialic acid by commercial exoneuraminidases results in an increase in the app...

Characterization of the kinetics of neural cell adhesion molecule homophilic binding

“…In these studies we show that NCAM homophilic binding can occur in the absence of heparin-sulphate proteoglycans and that heparin does not affect the amount of binding observed. The affinity constants determined for the interactions of adult and P0 NCAM are consistent with other reports which suggest that the rates of NCAM-mediated aggregation of brain vesicles or cells is inversely proportional to their sialic acid content [18][19][20]. The amount of PSA associated with NCAM decreases with postnatal development [19,21].…”

Polysialyltransferase expression is linked to neuronal migration in the developing and adult zebrafish

“…Therefore, recombinant EndoN enzyme, which selectively degrades polySia residues from NCAM (Rutishauser et al, 1985;Marx et al, 2001;Franz et al, 2005), was used to specifically remove PSA from the differentiating brain at 33 hpf, shortly after the onset of PSA-NCAM expression in the cerebellum (Marx et al, 2001). The efficiency of PSA removal in EndoN-and phosphate buffered saline (PBS) -treated control embryos was monitored by immunohistochemistry using the mAb735 anti-PSA antibody at 2 and 24 hr after injection.…”

Cardiac expression of polysialylated NCAM in the chicken embryo: Correlation with the ventricular conduction system

“…The removal of PSA from NCAM enhances the efficiency of NCAM homophilic binding (Hoffman and Edelman, 1983;Rutishauser et al, 1985). The influence of PSA on cell-cell interactions has also been shown in some cases to be independent of its effect on NCAM binding.…”