Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants

Abstract

Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease.

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

“…2A). The patterns of intracellular maturation and secretion for wild-type M and mutant Z AAT variants, measured for comparison, were consistent with those reported previously [24]. Quantification of the three variants using densitometry (n = 2) revealed kinetics of Trento intracellular N-glycan maturation and secretion efficiency only slightly lower than those of wild-type M AAT (Fig.…”

“…SERPINA1 alleles are expressed codominantly, and plasma levels of AAT in heterozygous patients are thought to result from the independent contributions of the two variants. However, hetero‐polymerization of the S and Z variants of AAT has been demonstrated by in vitro studies , and an example of heterotypic interactions has been reported in cells between Z and I (R39C) AAT . On this basis, we investigated whether Z AAT coexpression could exacerbate retention of Trento AAT, but this was found not to be the case.…”

“…Pulse‐chase experiments were performed as previously described . Twenty‐four hours after transfection, Hepa 1.6 cells were pulsed for 10 min with 35 S Met/Cys (EasyTag™ Express Protein Labelling mix; Perkin Elmer, Milan, Italy) and chased for 0, 15, 30, 60, 120 or 240 min.…”

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

“…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 .…”

Cysteines as Redox Molecular Switches and Targets of Disease

“…Cysteine mutations and aberrant disulfide bonding underlie the pathogenesis of CD40 deficiency (Lanzi et al, 2010), TNFR1-associated periodic fever syndrome (Lobito et al, 2006) and MiDY insulin-deficient diabetes (Liu et al, 2010). Mutations causing conformational alterations of alpha-1-antitrypsin make its only cysteine more prone to form aberrant disulfide bonds in the ER, thus facilitating the intracellular retention and polymerization of alpha-1-antitrypsin in Alpha-1-antitrypsin deficiency (AATD; Ronzoni et al, 2016). …”