Ordóñez Adriana, Pérez Juan, Tan Lu, Dickens Jennifer A, Motamedi-Shad Neda, Irving James A, Haq Imran, Ekeowa Ugo, Marciniak Stefan J, Miranda Elena, Lomas David A
*Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; Department of Cell Biology, Genetics and Physiology, University of Malaga, Malaga, Spain; Wolfson Institute for Biomedical Research, University College London, London, United Kingdom; and Department of Biology and Biotechnologies, "Charles Darwin," and Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University, Rome, Italy.
*Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; Department of Cell Biology, Genetics and Physiology, University of Malaga, Malaga, Spain; Wolfson Institute for Biomedical Research, University College London, London, United Kingdom; and Department of Biology and Biotechnologies, "Charles Darwin," and Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University, Rome, Italy
FASEB J. 2015 Jun;29(6):2667-78. doi: 10.1096/fj.14-267351. Epub 2015 Mar 10.
Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.
突变型Z α1-抗胰蛋白酶（E342K）在肝细胞内质网（ER）内聚集成聚合物，易引发肝脏疾病，而循环中低水平的Z α1-抗胰蛋白酶会因对中性粒细胞弹性蛋白酶抑制作用丧失而导致肺气肿。理想的治疗方法应在保留抑制活性的同时防止聚合物形成。在此，我们利用单克隆抗体技术来鉴定符合这两个要求的与Z α1-抗胰蛋白酶相互作用的分子。我们报告了一种单克隆抗体（4B12）的产生，它在体外以1:1的摩尔比阻断α1-抗胰蛋白酶的聚合，使对中性粒细胞弹性蛋白酶的抑制化学计量略有增加。基于单克隆抗体4B12的序列产生了一种单链可变片段（scFv）胞内抗体。ER内（scFv4B12KDEL）和分泌途径沿线（scFv4B12）的scFv4B12表达使Z α1-抗胰蛋白酶的细胞内聚合减少了60%。scFv4B12胞内抗体还增加了Z α1-抗胰蛋白酶的分泌，该分泌产物对中性粒细胞弹性蛋白酶仍保留抑制活性。单克隆抗体4B12识别一个可能位于螺旋A/C/G/H/I区域的不连续表位，其作用似乎是通过改变蛋白质动力学而非优先结合天然状态。这种新方法可能揭示小分子干预的新靶点，这些靶点可阻断向异常聚合物的转变，而不损害Z α1-抗胰蛋白酶的抑制活性。
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