Characterisation of a type II functionally-deficient variant of alpha-1-antitrypsin discovered in the general population

Abstract

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.

Intrahepatic heteropolymerization of M and Z alpha-1-antitrypsin

“…Unless otherwise specified, reagents for buffer preparation were from MilliporeSigma. Hexahistidine-tagged human A1AT introduced into the pQE-30 vector (QIAGEN) was expressed in the XL1-Blue strain of E. coli (Thermo Fisher Scientific), purified by nickel-affinity and ion-exchange chromatography, as described previously (43), and its purity was assessed by SDS-PAGE and nondenaturing PAGE. Plasma M, Z, and S A1AT were purified using Antitrypsin Select affinity resin (GE Healthcare) and ion-exchange chromatography, as described previously (44).…”

Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency

“…The Gly228 residue is located at the end of the F helix, and when replaced, the AT protein conformation is unstable, resulting in polymer formation, and thus, is not degraded and trapped in the cell [ 76 ]. Similarly, Arg229 is also on the F helix, and mutations at this position mostly affect the stability of the AT conformation, occasionally forming polymers that may be associated with slowing the rate of RCL insertion into the A-β-fold or disruption of the stability of AT-target protein complexes [ 77 – 79 ]. The heparin-induced conformational change may involve the C-terminal part of the AT molecule and not N-terminal sites alone [ 80 ], and Arg229* mutant can cause lack of C-terminal part of the AT molecule just right, which indicates that Arg229* variant may also affect functional domains HBS.…”

Evaluation of the diagnostic performance of Alpha-1-Antitrypsin in early detection of hepatocellular carcinoma

“…HCC has been linked to several sociodemographic factors such as significant male preponderance, which is probably due to a concentration of risk factors in males as well as variations in sex hormones (41). Recent findings show that specific SERPINA1 gene mutations induce A1AT proteins to have reduced anti-protease function (42,43). We also examined the subjects for the presence of S and Z-deficient A1AT alleles.…”