Functional characterization of amino acid variants of the reactive-center-loop of alpha-1-antitrypsin

Alpha-1-antitrypsin (AAT) is an abundant glycoprotein in the plasma, is synthetized in the liver and acts as an inhibitor of serine proteases like neutrophil elastase (HNE). Mutations in the AAT gene generally lead to reduced AAT levels in the plasma (AAT deficiency, AATD), thus diagnostic protocols are based primarily on AAT plasma quantification. The unbalance between AAT and the target proteases in the lungs predisposes to emphysema. AAT specifically binds HNE via its flexible and solvent exposed reactive center loop (RCL) and, upon cleavage, undergoes a conformational change that traps the protease in a covalent irreversible complex. Our aim was to identify amino acid variants in the RCL domain that impair anti-elastase activity. We considered AAT variants arising from all possible amino acid changes due to single nucleotide variations, excluding residues with high similarity. Interestingly, a subset of these variants is reported in the gnomAD database. The selected variants were expressed in HEK293T cells along with wild-type AAT, the common polymerogenic mutant Z (E342K) and the previously characterized dysfunctional Iners (G349R). We quantified AAT in conditioned media by ELISA and by non-denaturing PAGE analysis. In contrast with the Z AAT mutant, which is conformationally altered and preferentially accumulates in the endoplasmic reticulum as polymeric chains, all the selected RCL variants were secreted normally in the native monomeric conformation. Then anti-elastase activity of RCL variants was investigated by three independent assays: (1) analysis of HNE-AAT complexes by SDS-PAGE analysis; (2) binding to elastase adsorbed on multi-well plates, (3) inhibition of HNE activity on chromogenic substrates. Our results identified five fully dysfunctional AAT variants and four ones with diminished anti-elastase activity. Notably, due to their normal secretion, such variants would not be identified by AATD diagnostic protocols.