AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

Journal Article · Wed Mar 23 00:00:00 EDT 2016 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0151902· OSTI ID:1258608

Gaczynska, Maria ^[1]; Karpowicz, Przemyslaw ^[1]; Stuart, Christine E. ^[2]; Norton, Malgorzata G. ^[2]; Teckman, Jeffrey H. ^[3]; Marszal, Ewa ^[2]; Osmulski, Pawel A. ^[1]

---

1. Univ. of Texas Health Science Center at San Antonio, San Antonio, TX (United States). Dept. of Molecular Medicine
2. Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD (United States)
3. Saint Louis Univ. School of Medicine, St. Louis, Missouri (United States). Dept. of Pediatrics and Biochemistry

α₁-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography. PLOS

• Research Organization: Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE; Center for Biologics Evaluation and Research (CBER); U.S. Food and Drug Administration (CES)
• OSTI ID: 1258608
• Journal Information: PLoS ONE, Vol. 11, Issue 3; ISSN 1932-6203
• Publisher: Public Library of Science
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES