Characterization of the interaction between acetylcholinesterase and laminin : a template for discovering redundancy

[摘要] ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine,acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be ableto promote various non-cholinergic functions, including cell adhesion and neuriteoutgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouselaminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the siteon AChE recognised by certain monoclonal antibodies (MAbs) might be critical fordifferentiation. These MAbs were found to inhibit both laminin binding and cell adhesionin neuroblastoma cells. In this study, the structure and characteristics of this site wereinvestigated, using the AChE-laminin interaction as a template as well as a detailedepitope analysis of the MAbs. The interaction sites of AChE and laminin wereinvestigated using phage display, modelling and docking, synthetic peptides, enzymelinked immunosorbent assays (ELISAs) and conformational interaction site mapping.Docking of AChE with the single-chain variable fragments (scFvs) produced from thephage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Aspmotif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human)motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement ofthe AG-73 site (2719-2729). This site overlaps with laminin's heparin-binding site.Docking showed the major component of the interaction site on AChE to be the acidicArg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42,Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66.Epitope analysis showed the MAb's major recognition site to be the sequence Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerichsequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. These results define the interaction sites involved in the AChE-laminin interactionand suggest that the interaction plays a role in cell adhesion.Despite the in vitro demonstrations of the importance of AChE's non-classical functions,the AChE knockout survives. Results from this study suggest the possibility of functionalredundancy between AChE and other molecules in early development. Using these invitro findings that AChE is able to bind laminin-111, information on the interaction sites,as well as results from the monoclonal antibody (MAb) epitope analysis, the idea ofredundancy was investigated. Docking and bioinformatics techniques were used toinvestigate structurally similar molecules that have comparable spatiotemporal expressionpatterns in the embryonic nervous system. AChE has been shown to be involved in thepathogenesis of Alzheimer's disease, thus molecules associated with brain function andneurodegeneration were also investigated. Molecules with which AChE could be possiblyredundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoproteinreceptors and their variants. AChE was observed to dock with growth arrest-specificprotein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminininteraction. The AChE interaction site was shown to resemble the apolipoprotein-bindingsite on the low density lipoprotein receptor, and related molecules, including the lowdensity lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor(SORL1). These molecules, along with apoE, are associated with Alzheimer's disease.Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested;this is interesting as AChE has been implicated in both haematopoiesis andhaematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternativeligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, andalso suggested the existence of other binding partners.In conclusion, characterisation of the AChE-laminin interaction sites and investigation ofstructurally similar sites in other molecules suggests a role for AChE in the stabilizationof the basement membrane of developing neural cells and provides a feasible explanationfor the survival of the knockout mouse. Furthermore, the demonstrated similarity of theAChE interaction site to sites on molecules, notably the low density lipoprotein receptorfamily and SORL1 and their apolipoprotein ligands that are implicated in the pathologyof Alzheimer's disease, as well as the possible link to haematopoietic differentiation andcancers, warrants further investigation.

[发布机构] Stellenbosch University