Structural characterization of a homophilic binding site in the neural cell adhesion molecule (original) (raw)
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Neuroscience Letters, 1987
Recent studies have described the localization of functional and structural domains on the neural cell adhesion molecule NCAM. In the present study we have extended these observations to examine the location of the carbohydrate epitope recognized by the L2 monoclonal antibody. This carbohydrate moiety is localized to lhe 65,000-dalton amino-terminal fragment of NCAM (previously designated Frl), but is not present in the amino-terminal 25,000-dalton region of NCAM that contains the heparin-binding domain. We have also examined the role of this domain in NCAM-mediated cell adhesion, and have shown that incubation of an NCAM substratum with L2 monoclonal antibody inhibits cell attachment to this substratum. These data therefore suggest that the carbohydrate moiety recognized by L2 monoclonal antibody may be involved in the modulation of NCAM-mediated cell adhesion. The interaction between cell surface molecules is an integral component of cell recognition processes which underlie neural development. The use of immunological probes, particularly monoclonal antibodies, have proved useful for studying these processes during embryogenesis. Probably the best characterized cell adhesion molecule in the nervous system is the neural cell adhesion molecule NCAM [6, 18, 19]. Other neural CAMs have been identified and appear to regulate adhesive processes distinct from NCAM [8 10, 12, 13, 16]. Monoclonal antibodies have also been used to identify shared determinants on adhesion proteins, the most notable being a sulfated glucuronic acid-containing carbohydrate epitope [1, 22] recognized by the L2 and HNK-1 monoclonal antibodies which is present on a family of glycoproteins including the adhesion molecules NCAM, L l, J1, and myelin-associated glycoprotein [1 1, 12, 15, 16]. This carbohydrate moiety appears to be involved in neural cell adhe
Journal of Biological Chemistry, 2002
NCAM plays a key role in neural development and plasticity-mediating cell adhesion and differentiation mainly through homophilic binding. Until recently, attempts to modulate neuronal differentiation and plasticity through NCAM have been impeded by the absence of small synthetic agonists mimicking homophilic interactions of NCAM. We show here that a peptide, P2, corresponding to a 12-amino acid sequence localized in the FG loop of the second Ig module of NCAM, binds to the first Ig module, which is the natural binding partner of the second Ig module, with an apparent K d of 4.7 ؎ 0.9 ؋ 10 ؊6 M. P2 inhibits cell aggregation and induces neurite outgrowth from hippocampal neurons, maximal neuritogenic effect being obtained at a concentration of 0.8 M. The neuritogenic effect was inhibited by preincubation of P2 with the recombinant NCAM-IgI. Both the length of P2 and the basic amino acid residues at the N and C termini are important for its neuritogenic activity. Treatment of hippocampal cultures with P2 results in induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2. Thus, P2 is a potent mimetic of NCAM, and therefore, an attractive compound for the development of drugs for the treatment of neurodegenerative diseases.
Journal of Molecular Biology, 2008
The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 Å. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal β-strands are interchanged. β-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that β-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.
Homophilic and heterophilic binding activities of Nr-CAM, a nervous system cell adhesion molecule
The Journal of Cell Biology, 1992
Nr-CAM is a membrane glycoprotein that is expressed on neurons. It is structurally related to members of the N-CAM superfamily of neural cell adhesion molecules having six immunoglobulin-like domains and five fibronectin type III repeats in the extracellular region. We have found that the aggregation of chick brain cells was inhibited by anti-Nr-CAM Fab' fragments, indicating that Nr-CAM can act as a cell adhesion molecule. To clarify the mode of action of Nr-CAM, a mouse fibroblast cell line L-M(TK-) (or L cells) was transfected with a DNA expression construct encoding an entire chicken Nr-CAM cDNA sequence. After transfection, L cells expressed Nr-CAM on their surface and aggregated. Aggregation was specifically inhibited by anti-Nr-CAM Fab' fragments. To check the specificity of this aggregation, a fusion protein (FGTNr) consisting of glutathione S-transferase linked to the six immunoglobulin domains and the first fibronectin type III repeat of Nr-CAM was expressed in Esc...
Journal of Biological Chemistry, 1994
The neural cell adhesion molecule NCAM mediates intercellular adhesion by homophilic binding and its homophilic binding site has been mapped to a decapeptide sequence 243-KYSFNYDGSE-252 located within the third immunoglobulin-like domain of chick NCAM. To investigate the relationship between homophilic binding and NCAM-dependent neurite outgrowth, mutations were created in the binding site of NCAM-140 cDNA. Mutant NCAMs were expressed in L cells, and their ability to promote neurite outgrowth from chick retinal ganglion cells was assayed in coculture systems. Mutations that resulted in the loss of NCAM homophilic binding failed to promote neurite outgrowth from retinal cells. Alternatively, synthetic peptides containing the decapeptide sequence of the homophilic binding site were used to block NCAM homophilic interaction. Peptides that inhibited NCAM-NCAM binding also blocked neurite elongation. However, the peptide P5 (243-KYSFNYDGSELIIKKVDKSDE-263), despite being an inhibitor of N...
Polypeptide components and binding functions of neuron-glia cell adhesion molecules
Proceedings of the National Academy of Sciences, 1984
Neuron-glia cell adhesion molecule (Ng-CAM) has previously been shown to be present exclusively on neurons and to mediate adhesion between neuronal membranes and glial cells. In the present study, its chain structure, binding functions, and relation to N-CAM (the other known CAM on neurons) were investigated further.
Proceedings of the National Academy of Sciences, 2004
The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for seque...
Proceedings of the National Academy of Sciences, 1991
We have used a combination of hydropathy analysis of the intercellular adhesion molecule 1 (ICAM-1) sequence and dot-matrix comparison of the sequence with the homologous, but functionally distinct, protein myelin-associated glycoprotein to identify a putative functional binding region. One polar, and presumably surface-exposed, region of ICAM-1 showed no significant identity with myelin-associated glycoprotein. A synthetic peptide analog based on the sequence of this region (JF9) mimicked the inhibitory effects of the anti-ICAM-1 monoclonal antibody WEHI-CAM-1. These included inhibition of ICAM-1-dependent homotypic aggregation of Raji Burkitt lymphoma and phorbol-ester treated U937 cells at concentrations as low as 80 micrograms/ml (24 microM). In addition, at a concentration of 100 micrograms/ml, the peptide analog effectively inhibited cytotoxic cell activity, an ICAM-1-dependent effector function of the immune response. This simple method of sequence analysis may have general applicability to the identification of functional domains in homologous, but functionally distinct, proteins such as the translated products of gene families.