AFM imaging reveals topographic diversity of wild type and Z variant polymers of human α1-proteinase inhibitor

Maria Gaczynska , Przemysław Karpowicz , Christine E. Stuart , Malgorzata G. Norton , Jeffrey H. Teckman , Ewa Marszal , Pawel A. Osmulski

Abstract

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.
Author Maria Gaczynska
Maria Gaczynska,,
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, Przemysław Karpowicz (FCh / DBCh / LMCh)
Przemysław Karpowicz ,,
- Laboratory of Medical Chemistry
, Christine E. Stuart
Christine E. Stuart,,
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, Malgorzata G. Norton
Malgorzata G. Norton,,
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, Jeffrey H. Teckman
Jeffrey H. Teckman,,
-
, Ewa Marszal
Ewa Marszal,,
-
, Pawel A. Osmulski
Pawel A. Osmulski,,
-
Journal seriesPlos One, ISSN 1932-6203, (A 35 pkt)
Issue year2016
Vol11
No3
Pages1-19
Publication size in sheets0.9
DOIDOI:10.1371/journal.pone.0151902
URL https://doi.org/10.1371/journal.pone.0151902
Languageen angielski
LicenseJournal (articles only); published final; Uznanie Autorstwa (CC-BY); with publication
Not used for evaluationyes
Score (nominal)40
ScoreMinisterial score = 35.0, 28-09-2018, ArticleFromJournal
Ministerial score (2013-2016) = 40.0, 28-09-2018, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 2.806 (2) - 2016=3.394 (5)
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