Lee J, Novoradovskaya N, Rundquist B, Redwine J, Saltini C, Brantly M
Clinical Studies Section, Pulmonary-Critical Care Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1590, USA.
Mol Genet Metab. 1998 Apr;63(4):270-80. doi: 10.1006/mgme.1998.2680.
alpha 1-Antitrypsin (alpha 1AT) provides the major protection in the lung against neutrophil elastase-mediated proteolysis. Inheritance of alpha 1AT deficiency alleles is associated with an increased risk of emphysema and liver disease. alpha 1AT null alleles cause the total absence of serum alpha 1AT and represent the ultimate in a continuum of alleles associated with alpha 1AT deficiency. The molecular mechanisms responsible for absence of serum alpha 1AT include splicing abnormalities, deletion of alpha 1AT coding exons, and premature stop codons. We identified an Italian individual with asthma, emphysema, and a very low level of serum alpha 1AT. DNA sequencing demonstrated the Mprocida deficiency allele and a novel null allele, QOtrastevere (c654 G-->A, W194Z), a nonsense mutation near the intron 2 (IVS2) splice acceptor site. To determine the molecular basis of QOtrastevere and specifically to evaluate whether this nonsense mutation interfered with mRNA processing by altered splicing, we used a Chinese hamster ovary cell line permanently transfected with QOtrastevere or normal M alpha 1AT with and without IVS2. Northern blot analysis demonstrated that the normal M construct, with or without IVS2, expressed alpha 1AT mRNA of a similar size. The nonsense mutation was associated with moderately reduced alpha 1AT mRNA regardless of the presence or absence of IVS2. Reduction in alpha 1AT mRNA regardless of the opportunity for splicing supports a translational-translocation model as the cause of reduced alpha 1AT mRNA rather than the nuclear scanning model. Pulse-chase studies followed by immunoprecipitation demonstrated an endoplasmic reticulum-retained 31 kDa QOtrastevere alpha 1AT, which was rapidly degraded. Although mRNA content was moderately reduced, retention and rapid intracellular degradation of the truncated form are the major mechanisms for the absence of secreted alpha 1AT.
α1 -抗胰蛋白酶（α1AT）为肺部提供了针对中性粒细胞弹性蛋白酶介导的蛋白水解的主要保护作用。α1AT缺陷等位基因的遗传与肺气肿和肝病风险增加相关。α1AT无效等位基因导致血清α1AT完全缺失，代表了与α1AT缺陷相关的一系列等位基因中的极端情况。导致血清α1AT缺失的分子机制包括剪接异常、α1AT编码外显子的缺失以及过早的终止密码子。我们鉴定出一名患有哮喘、肺气肿且血清α1AT水平极低的意大利个体。DNA测序显示存在Mprocida缺陷等位基因以及一个新的无效等位基因QOtrastevere（c654 G→A，W194Z），这是一个位于内含子2（IVS2）剪接受体位点附近的无义突变。为了确定QOtrastevere的分子基础，特别是评估这种无义突变是否通过改变剪接干扰mRNA加工，我们使用了一种稳定转染了QOtrastevere或正常Mα1AT且有或无IVS2的中国仓鼠卵巢细胞系。Northern印迹分析表明，无论有无IVS2，正常的M构建体都表达大小相似的α1AT mRNA。无论有无IVS2，无义突变都与α1AT mRNA适度减少相关。无论有无剪接机会，α1AT mRNA的减少都支持翻译 - 转运模型是α1AT mRNA减少的原因，而非核扫描模型。脉冲追踪研究随后进行免疫沉淀，结果显示内质网滞留的31 kDa QOtrastevereα1AT，其迅速降解。尽管mRNA含量适度减少，但截短形式的滞留和细胞内快速降解是分泌型α1AT缺失的主要机制。
