Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM.

Abstract

Abstract. The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoelonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.

Neural Cell Adhesion Molecule (N-CAM) Homophilic Binding Mediated by the Two N-terminal Ig Domains Is Influenced by Intramolecular Domain-Domain Interactions

“…This form of Ig3 has been previously reported to inhibit bead and cell aggregation (18). However, the Ig3 peptide reported by Rao, et al to incorporate the proposed homophilic binding site and to inhibit N-CAM binding (P5) (16,17) exhibited no activity in this assay (Fig. 8A).…”

“…A number of Ig3-specific reagents have been reported to inhibit cellcell aggregation including antibodies, recombinant proteins, and peptides, and it has been proposed that self-association of the Ig3 domains is central to N-CAM binding (15)(16)(17)(18). However, we and others (3,19) have been unable to demonstrate self-association of the isolated Ig3 domain in solution.…”

“…Early electron microscopy studies on native N-CAM revealed a rod-shaped molecule with a flexible hinge (13,14), consistent with the predicted requirement for flexibility in cell adhesion molecules (1). A later series of experiments suggested that self-association of the third Ig domain, Ig3 was required for homophilic binding, and showed that a peptide, P5, whose sequence is contained within the Ig3 domain could inhibit cell attachment (15,16,17). Independent bead and cell-binding experiments also demonstrated self-association of Ig3.…”

Genomic organization of a novel glycosylphosphatidylinositol MAM gene expressed in human tissues and tumors

“…Based on the GPIM deduced protein, we postulate that this protein may be implicated in cell±cell interaction, as it contains several motifs (immunoglobulin-like domains, ®bronectin type III domain, MAM domain, GPI anchor) that are present among several adhesive molecules (Rao et al, 1992;Beckmann and Bork, 1993). The immunoglobulin superfamily, the largest class of CAMs, includes some well characterized GPI-anchored proteins.…”

Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity

“…For example, the first and second Ig domains interact with each other promoting neurite outgrowth (Thomsen et al, 1996;Kiselyov et al, 1997;Jensen et al, 1999;Rønn et al, 1999). The third Ig domain seems to bind to itself and is also capable of stimulating neurite outgrowth (Rao et al, 1992Sandig et al, 1994;Ranheim et al, 1996). The fourth Ig domain is involved in interaction with another NCAM, L1, and contains an alternatively spliced 10 amino acidlong sequence termed VASE, which when expressed abrogates the neurite outgrowth-promoting activity of NCAM (Small and Akeson, 1990;Saffell et al, 1994;Lahritz et al, 1997).…”