Homophilic adhesion mediated by the neural cell adhesion molecule involves multiple immunoglobulin domains.

Abstract

The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig

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

“…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.…”

“…Independent bead and cell-binding experiments also demonstrated self-association of Ig3. In addition, this study observed reciprocal interactions between the remaining Ig domains consistent with antiparallel binding of all 5 Ig domains (18). In contrast, solution and crystallographic studies have characterized an interaction between the N-terminal Ig domains Ig1 and Ig2 (3, 19 -21), and failed to detect any self-association of Ig3.…”

“…The monoclonal and domain-specific polyclonal antibodies were described previously (18). Fab fragments of protein G/A-purified antibodies were generated by papain cleavage (2 h at 37°C), and subsequently separated from the Fc fragments and any undigested antibody by a second protein G/A column.…”

“…The secreted proteins were purified from the media on protein A-Sepharose as described (23) and stored in aliquots at Ϫ80°C. Two recombinant Ig3 proteins with alternative N termini (Ig3, residues Lys 183 -Lys 288 (18), and Ig3s, residues Val 195 -Lys 288 (3)) were purified from E. coli as described previously. Recombinant proteins were quantified using the Bio-Rad DC assay employing BSA as a standard.…”

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).…”