Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides

Abstract

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

“…Bars represent means+SEM from four independent experiments. *p<0.05, **p<0.01 vs IL-1β essary for NCAM-induced neurite outgrowth [20]. This indicates that the ability of the mutated peptides to induce intracellular NCAM-signalling is abolished.…”

“…It has previously been reported that two or more alanine substitutions in the C3d peptide block its ability to induce neurite outgrowth and to increase intracellular Ca 2+ concentrations [20,24]. Furthermore, the capability of C3d to induce neurite outgrowth can be blocked by inhibitors of NCAM-induced intracellular signalling nec- C3d 3ala Fig.…”

“…C3d, a dendrimer of four peptide monomers (ASKKPKRNIKA) coupled to a lysine backbone, and two mutated C3d peptides-C3d 2ala (ASKKPAANIKA) and C3d 3ala (ASKAPAANIKA)-were produced and characterised as described previously [20]. The stated concentrations of peptides represent the concentrations of the peptide monomers (i.e.…”

“…NCAM signals through a Src kinase in both neurons and beta cells [32,41]. In neurons the NCAM-binding peptide C3d activates NCAM signalling [20] and we wished to examine in beta cells the effect of C3d on Src kinase activity. As seen in Fig.…”

“…The NCAM-140 isoform further interacts with the Src-homology kinase Fyn leading to Fak-recruitment and activation of the RasRaf-MEK-ERK pathway [19]. By means of combinatorial chemistry, a synthetic peptide termed C3d has been shown to bind NCAM [20]. C3d disrupts NCAM-mediated cell adhesion in vitro and induces intracellular signalling and responses similar to that activated by homophilic NCAM binding in neurons [21][22][23][24].…”