Hadfield, Lynsay Claire (2018) Heparin and heparin-like molecules inhibit the Alzheimer’s β-secretase (BACE1): considerations for biological assay and future therapeutic development. Doctoral thesis, Keele University.

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Abstract

The biologically and medically important heparan sulphate and heparin polysaccharides have previously been shown to modulate the activity of an aspartyl protease, β-secretase (BACE1), implicated in the aetiology of Alzheimer’s disease. Research groups investigating the activity of heparin with BACE1 have demonstrated both inhibitory and stimulatory effects of this glycosaminoglycan, and other analogues. In an attempt to understand this relationship, a review of the available recombinant BACE1 products was conducted to determine if protein purification tag had an effect on the heparin/heparan sulphate interaction with BACE1. FLAG-tagged BACE1 was identified as a suitable proxy for native, untagged BACE1 for activity studies.
In this study, the common purification tag, IgG Fc region, has been shown to interact with heparin and other glycosaminoglycans at acid pH (pH 4.0 and pH 5.0) with heparin affinity of > 800 mM. Further investigation, first of whole immunoglobulin (IgG & IgM) and then with Fab and F(ab’)2 antibody fragments, identified heparin binding at acid pH in all fragments tested. Thermal stability studies were conducted to identify heparin/HS structural requirements for this novel interaction, which suggest sulphate is necessary but not sufficient for activity.
Finally, a library of chemically sulphated non-heparin polysaccharides were utilised to identify ‘hit’ BACE1 inhibitors with beneficial BACE1 therapeutic attributes such as low molecular weight, minimal charge and attenuated off target effects, such as anti-coagulant activity. Differential scanning fluorimetry was identified as a potential high-throughput screening assay to replace fluorescence resonant energy transfer, during the initial nonheparin polysaccharide library BACE1 inhibitor screening.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Natural Sciences > School of Life Sciences
Contributors: Skidmore, MA (Thesis advisor)
Yates, Edwin (Thesis advisor)
Allin, Steven (Thesis advisor)
Depositing User: Lisa Bailey
Date Deposited: 08 Jun 2018 11:46
Last Modified: 20 Oct 2020 14:50
URI: https://eprints.keele.ac.uk/id/eprint/4998

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