Abstract:
Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neunte outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val2718-Arg-Lys-Arg-Leu 2722, Tyr2738-Tyr2739, Tyr2789-Ile- Lys-Arg-Lys2793 and Val2817-Glu-Arg-Lys2820, on the mouse laminin α1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg90-Glu-Leu-Ser- Glu-Asp95 on the omega loop, and also the involvement of Pro 40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61-Ala-Thr-Thr-Phe-Gln 66. Epitope analysis, using CLiPS™ technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu 84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Antibodies that recognized other features of the PAS area but not the Arg 90-Gly-Leu-Ser-Glu-Asp95 motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse. © The Authors.