Abstract
The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.
Highlights
Erophilic binding remains to be established, two heparan sulfate proteoglycans (HSPGs) with interesting developmental expression patterns, namely agrin and the 6C4 antigen [21,22,23], have been shown to serve as heterophilic receptors for neural cell adhesion molecule (NCAM) [15, 16, 24]
The absolute levels of cell binding should not be compared for the different substrates, but rather the relative changes in binding produced by specific perturbations, as featured in this study. To investigate whether these interactions were mediated by NCAM on the cells, the cells were treated with the appropriate anti-NCAM Fab antibody for 1 h and washed prior to the spot cell adhesion assay
The primary goals of this study were to evaluate a direct role for polysialic acid (PSA) in promotion of NCAM-mediated heterophilic cell adhesion and to characterize the molecular components and specificity of that adhesion
Summary
Erophilic binding remains to be established, two HSPGs with interesting developmental expression patterns, namely agrin and the 6C4 antigen [21,22,23], have been shown to serve as heterophilic receptors for NCAM [15, 16, 24]. The possibility of multiple NCAM binding activities presents a challenge to the interpretation of NCAM perturbation and gene mutation studies, in terms of molecular mechanism. One aspect of this ambiguity concerns the mode of action of the polysialic acid (PSA) moiety attached to NCAM. We have used the in vitro adhesion assay utilized by Storms et al [15, 16] to obtain direct evidence for a positive role for PSA in cell adhesion mediated by heterophilic binding of NCAM to two purified HSPG substrates, agrin and 6C4. A combination of specific antibody, enzyme, and competitive inhibitors (NCAM peptides and PSA fragments) was used to establish that this influence of PSA involves the known heparin-binding domain of NCAM and is distinct from the homophilic binding domain
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