Gao, Xin;
(2023)
Effect of the N-glycosylation of plasma proteins on their functional mechanism.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The glycosylation of immune proteins is a crucial aspect of their proper functionality, this thesis study the role of glycans in medically important proteins. Human Complement Factor H (CFH) is instrumental in regulating activated C3b to shield host cells. Comprising 20 short complement regulator (SCR) domains and eight N-glycosylation sites, CFH's N-terminal domains mediate C3b degradation while the C-terminal domains bind host cells for their protection. Our research on Pichia-generated CFH fragments has identified a self-association site at SCR-17/18, crucial for human factor H dimerization. The expression of SCR-17/18 in an E. coli system, devoid of glycans, revealed the absence of dimer formation. We further investigated this by purifying the full-length CFH and its C-terminal fragments from human plasma and Pichia pastoris, respectively, and enzymatically removing their glycans. Size-exclusion chromatography, mass spectrometry, and analytical ultracentrifugation were used to characterize their oligomeric states. Our results confirmed that glycans facilitate SCR-17/18 dimer formation. Affinity analysis of the crucial CFH-C3b interaction showed that deglycosylation decreases CFH's binding to C3b, highlighting the importance of glycosylation in CFH regulation. In parallel, I explored heavy-chain-only antibodies, specifically a potent anti-COVID-19 nanobody, C5, fused with the glycosylated Fc region of a human IgG1 antibody. This antibody can offer enhanced binding affinity, size advantages, and increased stability over conventional ones. X-ray and neutron scattering combined with Monte Carlo modelling helped determine the C5-Fc structure before and after deglycosylation. Notably, slight structural changes occurred after deglycosylation, indicating the significance of glycosylation in maintaining the C5-Fc solution structure. The Monte Carlo simulations generated realistic C5-Fc structures, with the top fits showing an array of flexible C5 conformations relative to the Fc region. The increased disorder post-deglycosylation emphasised the role of glycans in maintaining conformational stability. Overall this research uncovers previously unknown aspects of CFH regulatory relationships and the role of glycosylation in antibody stability.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Effect of the N-glycosylation of plasma proteins on their functional mechanism |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10180764 |
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