Atomic Force Microscopy reveals Diversity of Serpin Polymers

Abstract

Polymerization of a mutated form of antitrypsin is postulated to cause serpinopathy, a rare and incurable genetic disease. Antitrypsin is the canonical member of the serpin superfamily of structurally homologous proteins with metastable native structures. Most of serpins, including antitrypsin, are regulators of serine and cysteine protease cascades, and protect tissues from excessive actions of extracellular proteolytic enzymes. Polymerization of the mutant Z variant of antitrypsin results in the “loss of function” proteolytic damage of lungs, or in the “gain of function” degeneration of liver cells, loaded with toxic polymers. The understanding of polymerization mechanism holds the key to disease prevention, diagnostics and cures. However, metastability of serpins and complexity of their polymers makes structural studies a difficult task. There are several models of polymerization, based on in vitro studies and molecular modeling, however their relevance to in vivo polymerization and disease etiology has not been established. Here we employed atomic force microscopy (AFM) to compare topography of in vitro and in vivo formed antitrypsin polymers and oligomers. We found that commonly used in vitro methods to induce polymerization result in formation of topographically a very diverse population of oligomers. Most, if not all classes of the in vitro formed oligomers corresponded to similar classes of oligomers isolated from liver. The in vitro polymerization, however did not produce long strands of polymerized antitrypsin. The presence of such strands is the most striking feature in liver cells of human serpinopathy patients and in a mouse model. Here, we report results of our scanning probe microscopy structural analysis of long serpin polymers and propose the mechanism of their formation.