Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine,
acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able
to promote various non-cholinergic functions, including cell adhesion and neurite
outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse
laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site
on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for
differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion
in neuroblastoma cells. In this study, the structure and characteristics of this site were
investigated, using the AChE-laminin interaction as a template as well as a detailed
epitope analysis of the MAbs. The interaction sites of AChE and laminin were
investigated using phage display, modelling and docking, synthetic peptides, enzyme
linked immunosorbent assays (ELISAs) and conformational interaction site mapping.
Docking of AChE with the single-chain variable fragments (scFvs) produced from the
phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp
motif, 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 of
the 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 acidic
Arg90-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 prolinerich
sequences 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 interaction
and 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 functional
redundancy between AChE and other molecules in early development. Using these in
vitro 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 of
redundancy was investigated. Docking and bioinformatics techniques were used to
investigate structurally similar molecules that have comparable spatiotemporal expression
patterns in the embryonic nervous system. AChE has been shown to be involved in the
pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and
neurodegeneration were also investigated. Molecules with which AChE could be possibly
redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein
receptors and their variants. AChE was observed to dock with growth arrest-specific
protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin
interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding
site on the low density lipoprotein receptor, and related molecules, including the low
density 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 and
haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative
ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and
also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of
structurally similar sites in other molecules suggests a role for AChE in the stabilization
of the basement membrane of developing neural cells and provides a feasible explanation
for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the
AChE interaction site to sites on molecules, notably the low density lipoprotein receptor
family and SORL1 and their apolipoprotein ligands that are implicated in the pathology
of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and
cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy
klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle
vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in
amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes,
in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat
herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met
betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie,
sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die
struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin
interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop
het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese
peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie
area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting
varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat
die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro-
Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie
tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die
basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-
Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area
by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede
inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings
eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur
volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die
Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61
Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings
area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk
ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin
interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel
adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die
oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van
funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot
molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang.
Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie,
informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry
vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van
funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om
molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese
senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so
moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele
oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican;
perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante.
Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien
E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te
betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die
apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos
die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde
reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende
rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die
verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van
belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en
haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin
interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met
identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol
vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige
verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van
die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met
Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese
differensiering en kanker, lê die grondslag vir verdere ondersoeke.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20132 |
| Date | 03 1900 |
| Creators | Swart, Chrisna |
| Contributors | Johnson, Glynis, Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 247 p. : ill. |
| Rights | Stellenbosch University |
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