The pathological Trento variant of alpha‐1‐antitrypsin (E75V) shows nonclassical behaviour during polymerization

Abstract

Severe alpha‐1‐antitrypsin deficiency (AATD) is most frequently associated with the alpha‐1‐antitrypsin (AAT) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early‐onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD, typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. Polyacrylamide gel electrophoresis (PAGE) and ELISA‐based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localized between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilizing interaction between helix C and the posthelix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT.

Real-world clinical applicability of pathogenicity predictors assessed onSERPINA1mutations in alpha-1-antitrypsin deficiency

“…Formation of polymers both in cell extracts (Figure A) and culture media (Figure B) was analyzed by native PAGE. Intracellular and secreted Z α1AT was overwhelmingly polymeric, consistently with previous in vitro and in vivo studies (Fra et al., , ; Miranda et al., ; Tan et al., ). The expression of p.P313S, p.P279T, and p.G282R variants showed polymer/monomer proportions similar to Z α1AT (Figure A).…”

“…Vectors encoding for the α1AT variants were obtained by site‐directed mutagenesis of M1V (Medicina et al., ) using the QuikChange II Mutagenesis Kit (Agilent) with primers listed in . HEK293T/17 (ATCC#CRL‐11268) or Hepa1‐6 cells (ATCC#CRL‐1830) were maintained in DMEM‐10% FBS (Sigma) and transfected by PEI "Max" (Polysciences Inc.) or Lipofectamine2000 (Thermo Fisher) as previously described (Miranda et al., ; Ronzoni et al., ). Twenty‐four hours after transfection, we collected the cell media and lysed the cells in 1% NP40/20 mM Tris–HCl pH 7.4/150 mM NaCl/10 mM N‐ethylmaleimide/protease inhibitors, then discarding nuclei by 30′ centrifugation at 800 g .…”

“…Quantification of α 1 ‐AT in culture media was performed by sandwich ELISA as described (Miranda et al., ). Briefly, 96‐well plates (Costar 3590) were coated with rabbit polyclonal anti‐α 1 AT (DAKO; 2 μg/mL) and saturated for 1 hr at 37°C with a blocking buffer (PBS, 0.25% BSA).…”

“…were maintained in DMEM-10% FBS (Sigma) and transfected by PEI "Max" (Polysciences Inc.) or Lipofectamine2000 (Thermo Fisher) as previously described (Miranda et al, 2017;Ronzoni et al, 2016).…”

“…Those studied in more detail are polymerogenic mutants, such as M(malton) (p.F75del) (Graham et al, 1989), S(iiyama) (p.S77F) (Lomas, Finch, Seyama, Nukiwa, & Carrell, 1993), and King's (p.H358D) (Miranda et al, 2010). Others such as the I (p.R63C) (Graham et al, 1989;Ronzoni et al, 2016), Queen's (p.K178N) (Nyon et al, 2012), Baghdad (p.A360P) (Haq et al, 2016), Trento (p.E99V) (Miranda et al, 2017), and P(brescia) (p.G249R) (Medicina et al, 2009) are associated with milder polymerogenicity and minor plasma deficiency. The circulating deficiency of 1AT may be exacerbated by supervening reduced activity due to decreased binding affinity and inhibitory capacity against elastase, as reported for Z, F (p.R247C), Queen's and Baghdad mutations (Cook, Burdon, Brenton, Knight, & Janus, 1996;Haq et al, 2016;Nyon et al, 2012;Okayama, Brantly, Holmes, & Crystal, 1991).…”