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Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants

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Abstract

Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional ‘type-2’ variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT. This has potential clinical relevance: in addition to the dysfunctional Pittsburgh and Iners variants reported previously, several uncharacterized RCL variants are present in genome variation databases. To prospectively evaluate the impact of RCL variations on secretion and anti-protease activity, here we performed a systematic screening of amino acid substitutions occurring at the AAT–NE interface. Twenty-three AAT variants that can result from single nucleotide polymorphisms in this region, including 11 present in sequence variation databases, were expressed in a mammalian cell model. All demonstrated unaltered protein folding and secretion. However, when their ability to form stable complexes with NE was evaluated by western blot, enzymatic assays, and a novel ELISA developed to quantify AAT–NE complexes, substrate-like and NE-binding deficient dysfunctional variants were identified. This emphasizes the ability of the RCL to accommodate inactivating substitutions without impacting the integrity of the native molecule and demonstrates that this class of molecule violates a generally accepted paradigm that equates circulating levels with functional protection of lung tissue.

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Supporting data are included as electronic supplementary material.

Abbreviations

AAT:

Alpha-1-antitrypsin

AATD:

Alpha-1-antitrypsin deficiency

ELISA:

Enzyme-linked immunosorbent assay

NE:

Neutrophil elastase

PPE:

Porcine pancreatic elastase

RCL:

Reactive center loop

SI:

Stoichiometry of inhibition

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Acknowledgements

The authors thank Kaoutar Loukili and Giulia Bartoli for sharing reagents and protocols, Professor Fabrizio Gangemi (University of Brescia, Brescia, Italy) and Professor David A Lomas (UCL, London, UK) for insightful discussions of the work.

Funding

This work was mainly supported by a grant from the Alpha-1 Foundation USA (ID:829920) to AF. JAI is supported by the Medical Research Council UK (MR/V034243/1) and the Alpha-1 Foundation (ID: 1036784). EM was supported by the Alpha-1 Foundation (ID: 497841).

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Authors and Affiliations

  1. Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

    Andrea Denardo, Emna Ben Khlifa, Mattia Bignotti, Roberta Giuliani & Annamaria Fra

  2. Department of Biology and Biotechnologies ‘Charles Darwin’, Sapienza University of Rome, Rome, Italy

    Emanuela D’Acunto & Elena Miranda

  3. UCL Respiratory and the Institute of Structural and Molecular Biology, University College London, London, UK

    James A. Irving

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  1. Andrea Denardo
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Contributions

AD and AF designed the research project; AD, EBK, MB, RG, EM, ED, and AF performed experiments; AD and JAI performed structural analysis; AD, EBK, MB, EM, JAI, and AF analyzed data; AD, JAI, and AF wrote the paper. All authors read and approved the manuscript.

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Correspondence to Annamaria Fra.

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Denardo, A., Ben Khlifa, E., Bignotti, M. et al. Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants. Cell. Mol. Life Sci. 81, 6 (2024). https://doi.org/10.1007/s00018-023-05059-1

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