Point mutation of a non-elastase-binding site in human α1-antitrypsin alters its anti-inflammatory properties

Yotam Lior, Mariana Zaretsky, David E. Ochayon, Diana Lotysh, Boris M. Baranovski, Ronen Schuster, Ofer Guttman, Amir Aharoni, Eli C. Lewis

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo. Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo. Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding. Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

Original languageEnglish
Article number759
JournalFrontiers in Immunology
Volume9
Issue numberMAY
DOIs
StatePublished - 1 May 2018

Keywords

  • Anti-inflammatory
  • Inflammation
  • Pharmacokinetics
  • Protein structure
  • Reactive center loop
  • Recombinant protein
  • α1-antitryspin

Fingerprint

Dive into the research topics of 'Point mutation of a non-elastase-binding site in human α1-antitrypsin alters its anti-inflammatory properties'. Together they form a unique fingerprint.

Cite this