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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.

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a β-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a β-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state., Competing Interests: The authors declare no conflict of interest.

Background: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern., Methods: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy., Results: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation., Conclusions: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein., General Significance: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development., (© 2013.)