Induction of Neuronal Differentiation by a Peptide Corresponding to the Homophilic Binding Site of the Second Ig Module of the Neural Cell Adhesion Molecule

Abstract

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.The neural cell adhesion molecule (NCAM) 1 is a cell surface glycoprotein that belongs to the Ig superfamily. NCAM is primarily expressed in neurons, glial cells, and skeletal muscle. Alternative splicing of mRNA transcribed from a single gene generates three major NCAM isoforms: NCAM-A (180 kDa), NCAM-B (140 kDa), and NCAM-C (120 kDa). These isoforms have identical extracellular parts consisting of five N-terminal Ig-like modules (IgI-IgV) followed by two fibronectin type III modules (1). The transmembrane isoforms, NCAM-A and NCAM-B, have intracellular parts of different sizes, whereas NCAM-C is linked to the cell membrane via a glycosylphosphatidylinositol anchor. NCAM is known to mediate Ca 2ϩ -independent cell-cell and cell-substratum adhesion via homophilic (NCAM binding to NCAM) and heterophilic (e.g. NCAM binding to heparin/heparan sulfate proteoglycans) interactions, respectively (2-4). NCAM homophilic binding was originally reported to depend on a reciprocal interaction involving IgIII modules of two opposing NCAM molecules (5, 6). Later, an interaction involving all five Ig modules was also suggested (7). A double reciprocal binding between the recombinant IgI and IgII modules was demonstrated by means of surface plasmon resonance analysis (8), and recently, the three-dimensional structure of the IgI and IgII modules of rat NCAM was determined by both NMR spectroscopy and x-ray crystallography, resulting in the identification of homophilic binding sites in the IgI and IgII modules (9, 10).Neurons have been shown to respond with an increased neurite outgrowth to both glycosylphosphatidylinositol-linked and transmembrane isoforms of NCAM expressed on the surface of non-neuronal cells (11). NCAM homophilic binding has been suggested to activate the fibroblast g...

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

“…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]. C3d has recently been reported to protect neurons against apoptosis via PI3K and Akt activation [18].…”

The metastasis-promoting S100A4 protein confers neuroprotection in brain injury

“…Rat hippocampal neurons (E19) were prepared according to Soroka et al 53 After seven days in vitro (DIV), neurons were treated with S100A4, H3 or H6 for 1 h followed by KA (300 mM) or H2O2 (60 mM, both from Sigma). Inhibitory antibodies to IL-10R (R&D Systems) or the JAK inhibitor (Calbiochem) were added 30 min before S100A4.…”

“…Hippocampal neurons were stimulated with peptides, grown for 24 h and stained with Coomassie Blue R250. Neurite outgrowth was evaluated as described in Soroka et al 53 Phosphorylation of Akt, CREB and ERK was determined by phosphospecific antibody cell-based enzyme-linked immunosorbent assay (ELISA) as the ratio of phosphorylated to total protein 56 .…”

Peptide Mimetic of the S100A4 Protein Modulates Peripheral Nerve Regeneration and Attenuates the Progression of Neuropathy in Myelin Protein P0 Null Mice

“…The cells were stimulated with the peptide or S100A4 and grown for 24 h. To evaluate neurite outgrowth, the cultures were stained with polyclonal rabbit antirat growth-associated protein-43 antibodies and analyzed by using computerassisted microscopy as described previously (34).…”

“…Rat hippocampal neurons (E19) were isolated as previously described (34) and plated at a density of 5,000 cells per well in eight-well LabTek Permanox slides (NUNC, Roskilde, Denmark) coated with laminin (5 μg/mL; Sigma-Aldrich). The cells were stimulated with the peptide or S100A4 and grown for 24 h. To evaluate neurite outgrowth, the cultures were stained with polyclonal rabbit antirat growth-associated protein-43 antibodies and analyzed by using computerassisted microscopy as described previously (34).…”