Structural characterisation of tissue-derived, disease-associated polymers of alpha-1-antitrypsin using conformation-selective antibodies and single-particle reconstructions from electron microscopy images

α1-Antitrypsin is an abundant plasma inhibitor of neutrophil elastase,expressed at high levels by hepatocytes, and one of the causative agents of a class of conformational diseases termed serpinopathies. In its active state, α1-antitrypsin is in a kinetically stable, but thermodynamically unstable, configuration, rendering it susceptible to inappropriate conformationalchange. In individuals homozygous for the Z (E342K) mutation, α1-antitrypsin accumulates in the liver as dense intracellular deposits, leading to a reduced level in circulation. These deposits are the consequence of an ‘ordered aggregation’ that yields linear, unbranched protein chains, termed polymers, that are both extremely stable and functionally inactive. The circulating deficiency results in a protease-antiprotease imbalance in the lung, predisposing affected individuals to emphysema and COPD, whilst the hepatic accumulation can lead to liver disease, including cirrhosis and hepatocellular carcinoma. Our aim is to define the molecular details of the polymerisation pathway, in which α1-antitrypsin passes through different conformational states as it transitions from the active monomer via one or more structural intermediates to a hyperstable polymeric form. Different models have been proposed for the terminal structure adopted by the pathological polymer; these are are largely based on characterisation of polymers produced under conditions mechanistically or biologically distinct from those existing in vivo, and as such their relevance to the pathological context has not been established. To probe the structural and energetic aspects of the polymerisation pathway, we have generated a molecular toolkit of conformation-specific monoclonal antibodies (mAbs), and mapped their epitopes. We have utilised these mAbs and applied single-particle reconstruction techniques to negative stain and cryo-EM images of polymers extracted from patient explant liver tissue. The resulting maps, in conjunction with molecular modelling, have allowed us to critically evaluate the proposed mechanisms of polymer formation.