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Structure and function studies of lipid binding to zinc-α₂-glycoprotein

Zahid, Henna; (2020) Structure and function studies of lipid binding to zinc-α₂-glycoprotein. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Zinc-α2-glycoprotein (ZAG) is a 42kDa ubiquitously expressed adipokine, with a major histocompatibility complex class I protein fold. ZAG has three domains α1, α2 and α3; it regulates body fat mass and is associated with cachexia and obesity. Despite structural and biochemical observations that ZAG binds lipids, its intrinsic ligand is still unknown. ZAG however binds the dansylated C11 fatty acid DAUDA and the boron dipyrromethene fatty acid C16-BODIPY in its groove between its α1 and α2 helices. To determine if there were one or two fatty acid binding sites in the ZAG groove, conserved residues in the groove were subjected to site-directed mutagenesis. Lipid binding fluorescent studies of the resulting mutants revealed that the ZAG groove has two clearly different binding sites for the DAUDA and C16-BODIPY probes. To investigate whether the α3 domain influences the lipid-binding groove in the α1 and α2 domains, the crystal structure of the α1:α2 domains only was solved to a resolution of 2.6 Å, and accompanied with binding studies. Intriguingly, the structure and lipid binding function of the α1:α2 domains was retained, confirming that the α3 domain is not directly relevant for lipid-binding in ZAG. Further evidence to support multiple sites in the ZAG groove was provided by multiwavelength analytical ultracentrifugation that showed that ZAG accommodated both DAUDA and C16-BODIPY in its groove at equimolar concentrations. At higher lipid concentrations, ZAG formed dimers and trimers. Small angle X-ray scattering confirmed that these ZAG complexes showed increases in RG and length (L) with the addition of excess DAUDA and C16-BODIPY. Steady state fluorescent displacement assays showed that ZAG has a wider lipid-binding repertoire than thought, which included ceramide and phospholipids in addition to long-chain polyunsaturated fatty acids. In conclusion, the above findings clarified the molecular basis of lipid binding to ZAG by showing that ZAG has a wide range of expression and binding function in secretory tissues, to allow it to exert a tissue-specific function.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structure and function studies of lipid binding to zinc-α₂-glycoprotein
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/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
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 Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10093850
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