Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.
UCL Respiratory and the Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK.
Hum Mol Genet. 2018 May 15;27(10):1785-1793. doi: 10.1093/hmg/ddy090.
The most common genotype associated with severe α-1-antitrypsin deficiency (AATD) is the Z homozygote. The Z variant (Glu342Lys) of α-1-antitrypsin (AAT) undergoes a conformational change and is retained within the endoplasmic reticulum (ER) of hepatocytes leading to the formation of ordered polymeric chains and inclusion bodies. Accumulation of mutated protein predisposes to cirrhosis whilst plasma AAT deficiency leads to emphysema. Increased risk of liver and lung disease has also been reported in heterozygous subjects who carry Z in association with the milder S allele (Glu264Val) or even with wild-type M. However, it is unknown whether Z AAT can co-polymerize with other AAT variants in vivo. We co-expressed two AAT variants, each modified by a different tag, in cell models that replicate AAT deficiency. We used pull-down assays to investigate interactions between co-expressed variants and showed that Z AAT forms heteropolymers with S and with the rare Mmalton (Phe52del) and Mwurzburg (Pro369Ser) mutants, and to a lesser extent with the wild-type protein. Heteropolymers were recognized by the 2C1 mAb that binds to Z polymers in vivo. There was increased intracellular accumulation of AAT variants when co-expressed with Z AAT, suggesting a dominant negative effect of the Z allele. The molecular interactions between S and Z AAT were confirmed by confocal microscopy showing their colocalization within dilated ER cisternae and by positivity in Proximity Ligation Assays. These results provide the first evidence of intracellular co-polymerization of AAT mutants and contribute to understanding the risk of liver disease in SZ and MZ heterozygotes.
与严重的α-1-抗胰蛋白酶缺乏症（AATD）相关的最常见基因型是 Z 纯合子。α-1-抗胰蛋白酶（AAT）的 Z 变体（Glu342Lys）发生构象变化，并在内质网（ER）中保留，导致有序聚合链和包含体的形成。突变蛋白的积累易导致肝硬化，而血浆 AAT 缺乏导致肺气肿。携带 Z 与轻度 S 等位基因（Glu264Val）甚至与野生型 M 相关的杂合子也报告了肝脏和肺部疾病的风险增加。然而，目前尚不清楚 Z AAT 是否可以在体内与其他 AAT 变体共同聚合。我们在复制 AAT 缺乏的细胞模型中共同表达两种 AAT 变体，每种变体都经过不同的标签修饰。我们使用下拉测定法研究共表达变体之间的相互作用，并表明 Z AAT 与 S 以及罕见的 Mmalton（Phe52del）和 Mwurzburg（Pro369Ser）突变体形成杂聚物，并且与野生型蛋白的程度较小。杂聚物被 2C1 mAb 识别，该 mAb 在体内与 Z 聚合物结合。当与 Z AAT 共同表达时，AAT 变体的细胞内积累增加，表明 Z 等位基因具有显性负效应。共聚焦显微镜显示 S 和 Z AAT 之间的分子相互作用证实了它们在扩张的内质网腔中的共定位，并通过接近连接测定法呈阳性。这些结果首次提供了 AAT 突变体在细胞内共同聚合的证据，并有助于理解 SZ 和 MZ 杂合子中肝脏疾病的风险。
