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, 14 (1), e0206955

Characterisation of a Type II Functionally-Deficient Variant of alpha-1-antitrypsin Discovered in the General Population


Characterisation of a Type II Functionally-Deficient Variant of alpha-1-antitrypsin Discovered in the General Population

Mattia Laffranchi et al. PLoS One.


Lung disease in alpha-1-antitrypsin deficiency (AATD) results from dysregulated proteolytic activity, mainly by neutrophil elastase (HNE), in the lung parenchyma. This is the result of a substantial reduction of circulating alpha-1-antitrypsin (AAT) and the presence in the plasma of inactive polymers of AAT. Moreover, some AAT mutants have reduced intrinsic activity toward HNE, as demonstrated for the common Z mutant, as well as for other rarer variants. Here we report the identification and characterisation of the novel AAT reactive centre loop variant Gly349Arg (p.G373R) present in the ExAC database. This AAT variant is secreted at normal levels in cellular models of AATD but shows a severe reduction in anti-HNE activity. Biochemical and molecular dynamics studies suggest it exhibits unfavourable RCL presentation to cognate proteases and compromised insertion of the RCL into β-sheet A. Identification of a fully dysfunctional AAT mutant that does not show a secretory defect underlines the importance of accurate genotyping of patients with pulmonary AATD manifestations regardless of the presence of normal levels of AAT in the circulation. This subtype of disease is reminiscent of dysfunctional phenotypes in anti-thrombin and C1-inibitor deficiencies so, accordingly, we classify this variant as the first pure functionally-deficient (type II) AATD mutant.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Inhibitory mechanism of alpha-1-antitrypsin.
A schematic illustrating the inhibitory mechanism of alpha-1-antitrypsin (AAT), showing the progression from active inhibitor (I, PDB: 1QLP) and active protease (E, PDB: 1OPH), to the formation of the reversible Michaelis complex (E-I, PDB: 1OPH), and the branched pathway that leads to irreversible complex formation (EI*, PDB: 2D26) or cleaved inhibitor (Ic, PDB: 1EZX) and active enzyme (E). The figure was prepared with PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
Fig 2
Fig 2. Glycine 349 localization and conservation within the alpha-1-antitrypsin reactive centre loop.
(A) The Met358 (P1) and Ser359 (P1’) residues, critical for the anti-protease activity of AAT, are shown as black sticks on the native AAT structure (PDB: 1QLP), while the residue Gly349 (P10) is indicated by a blue stick. β-sheet A is blue, β-sheet B is green and β-sheet C is yellow; the RCL is coloured in red. The figure was prepared with PyMOL. (B) Conservation of residues in the RCL (top sequence, from residue G344 to P362) is represented using WebLogo [56], calculated from a sequence alignment of the SERPINA1 orthologues or from human serpins paralogues.
Fig 3
Fig 3. Characterization of the G349R variant in cell models.
(A) AAT levels in cell media from transfected HEK293T or Hepa 1–6 cells were quantified by sandwich ELISA and represented as percentages of wild-type M levels (mean ± SD, n = 3; one-way ANOVA, p < 0.0001; two-tailed unpaired t-test between each variant and M AAT, n.s non-statistically significant, **p < 0.001, ***p < 0.0001). (B) Immunoblots with anti-total AAT pAb loaded with equal volume of cell media from HEK293T (left) or Hepa 1–6 (right) cells expressing the indicated variants, resolved by 7.5% w/v acrylamide SDS-PAGE (top) and 8% Native-PAGE (bottom). (C) Immunoblots with anti-total AAT pAb of NP40-soluble (SOL) or insoluble (INS) cellular fractions from HEK293T (left) or Hepa 1–6 (right) cells expressing the indicated variants, resolved by 7.5% SDS-PAGE.
Fig 4
Fig 4. The AAT G349R variant has a defective inhibitory mechanism.
(A) Cell media of HEK293T transfected cells were incubated with (+) or without (–) equimolar or double concentrations of neutrophil elastase (HNE) for 30 min at 37°C, and the complexes (68 kDa, dark arrow) were resolved from unreacted AAT monomers (52 kDa) or cleaved forms (48 kDa, white arrow) by 7.5% w/v acrylamide SDS–PAGE and immunoblot with anti-AAT pAb. (B) An enzymatic assay using the pSuccAla3 chromogenic substrate with porcine pancreatic elastase (PPE) that had been preincubated with an increasing ratio of either wild-type M1V (dashed line) or Iners (dotted line) AAT variants from HEK293T cell media. pcDNA (solid line) represents the media of HEK293T cells not expressing AAT (n = 2, one-way ANOVA, p-value < 0.017). (C) Relative protease activity in the presence of increasing ratios of recombinant AAT wild-type M1V (solid line) or G349R variants (dashed line). The upper panel summarises the experiments using HNE, while the lower panel shows the inhibition of chymotrypsin (CHT) (n = 2). (D) Recombinant AAT wild-type M1V and G349R variants were incubated with (+) or without (–) an equimolar concentration of human purified plasma kallikrein (KLK) for 30 min at 37°C, and the samples were resolved by 4–12% w/v acrylamide SDS–PAGE. Arrows point to the uncleaved (48 kDa, dark arrow) and the cleaved forms (44 kDa, white arrow).
Fig 5
Fig 5. Comparative structural analysis and molecular dynamics simulations suggest an impaired presentation and insertion of the RCL.
(A) Residues in the vicinity of position 349 (yellow) following loop insertion are shown as sticks for cleaved wild-type alpha-1-antichymotrypsin (PDB: 2ACH, purple), the A349R variant (PDB: 1AS4, red) and AAT (PDB: 1EZX, black). Red arrows show the compensatory side-chain movements that occur in alpha-1-antichymotrypsin to accommodate the bulky charged arginine residue. The figure was prepared using PyMol. (B) Probability distribution of the angle φ defined in the text, as calculated in MD simulations for the wild type (solid line) and the G349R variant (dashed line). (C) Average number of hydrogen bonds between residue 349 and selected residues calculated in the MD simulations, for the wild-type AAT (wt, black) and the Iners variant (G349R, grey).

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