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

Denardo, Andrea; Ben Khlifa, Emna; Bignotti, Mattia; Giuliani, Roberta; D’Acunto, Emanuela; Miranda, Elena; Irving, James A; (2024) Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants. Cellular and Molecular Life Sciences , 81 (1) , Article 6. 10.1007/s00018-023-05059-1.

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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.

Type: Article
Title: Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants
Location: Switzerland
DOI: 10.1007/s00018-023-05059-1
Publisher version: http://dx.doi.org/10.1007/s00018-023-05059-1
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher's terms and conditions.
Keywords: SERPINA1, Alpha-1-antitrypsin deficiency, Neutrophil elastase, Type-2 alpha-1-antitrypsin deficiency, Molecular dysfunction, Protease inhibitor
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Respiratory Medicine
URI: https://discovery.ucl.ac.uk/id/eprint/10186069
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