Role of the α3 and α1α2α3 domains of zinc α2 glycoprotein in health and obesity

Abstract

Zinc α2 glycoprotein (ZAG) is a 42kDa adipokine with a class I MHC fold containing three domains; α1, α2 and α3. Fatty acids bind to its α1α2 groove. ZAG expression is inversely related to body fat mass and ZAG regulates lipolysis through an unknown mechanism. The ZAG: fatty acid crystal structure revealed ZAG tetramers and an unexpected α3 fatty acid binding site. The first aim of this thesis was to express recombinant E. coli ZAG α3 to investigate fatty acid binding using fluorescence, and tetramer formation using microfluidic diffusional sizing (MDS) in physiological conditions. Attempts at expressing and purifying α3 protein were not successful. A pET23a-α3 construct inhibited E. coli growth. pGEX-2TK GST–α3 and pGEX-2TK GST-3–HIS constructs expressed soluble proteins in E. coli, but the proteins degraded. A pET16b construct containing ZAG with an engineered thrombin cleavage site between its α1α2 and α3 domains produced inclusion bodies but refolded incorrectly. A second aim of this thesis was to optimise the use of the new MDS based Fluidity One-M instrument by measuring the affinity of Zagy-1 antibody binding to recombinant E.coli ZAG. The Zagy-1: ZAG affinity (KD) was 12 pM. This affinity was unaffected by ZAG denaturation, ionic strength and presence of fatty acid. The Zagy-1 antibody was used to measure ZAG concentrations in human plasma from sixteen obese patients’ pre and 9-months post Roux-En-Y surgery by ELISA. Patients underwent significant weight loss (p=<0.0001) and the concentration of ZAG increased after Roux-En-Y surgery (p=0.0684), demonstrating its role in body mass regulation.