Glutamyl-asparagine

The two major isoforms (180 kDa and 140 kDa) of the neural cell adhesion molecule (N-CAM) are crucially involved in neurogenesis and brain repair via activation of the mitogen-activated protein kinase (MAPK) cascade. Modification by glycosylation, and homophilic and heterophilic interactions regulate the function of N-CAM, but little is known about the interplay of these processes. In the neuron-like PC12 cell line, extracellular small acidic peptides have been shown to modulate the expression of N-CAM mRNA and protein and regulate its translocation to the plasma membrane. Among these peptides, a synthetic Ig-III-like short sequence (H2N-DDSDEEN-COOH), designated sSP, was particularly potent. In this study, we analyzed the cross-talk between nerve growth factor (NGF) and extracellular sSP in native and N-CAM-transfected PC12 cells to determine if these systems interact to modulate transduction pathways and regulate early steps of neurogenesis in vitro. Our results indicate that sSP accelerated the phosphorylation of extracellular regulated kinase-1 (ERK1) and -2 (ERK2) and promoted plasma membrane translocation of 180 kDa N-CAM. By stabilizing cell-cell contacts and promoting cell cluster formation, these events, which were mediated via a significant increase in intracellular Ca2+, regulated some of the early stages of the NGF-induced differentiation process.

DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide-N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.