Vest Katherine E, Apponi Luciano H, Banerjee Ayan, Pavlath Grace K, Corbett Anita H
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA.
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA.
J Neuromuscul Dis. 2015 Oct 20;2(4):439-446. doi: 10.3233/JND-150111.
Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs.
We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1.
We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD.
The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD.
This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.
眼咽型肌营养不良症（OPMD）是一种影响特定骨骼肌的迟发性疾病，由编码mRNA加工蛋白聚腺苷酸结合蛋白核1（PABPN1）的基因中的（GCG）n扩展突变引起。PABPN1中的扩展导致PABPN1蛋白中N端丙氨酸残基的一段序列从正常的10个增加到12 - 18个。鉴于这种适度的变化，如果不使用标记构建体，就无法检测到突变蛋白。
我们试图生成一种多克隆抗体，该抗体能够识别丙氨酸扩展型而非野生型PABPN1，目的是能够分析丙氨酸扩展型PABPN1的表达和定位。
我们用一种GST标记的丙氨酸肽免疫兔子，并针对在细胞培养物以及OPMD动物模型中表达的丙氨酸扩展型PABPN1检测所得血清。
所得的α - 丙氨酸抗体检测到含有14个或更多丙氨酸残基的PABPN1蛋白。重要的是，α - 丙氨酸抗体可用于检测来自OPMD小鼠或果蝇模型肌肉中的丙氨酸扩展型PABPN1。
这种α - 丙氨酸抗体提供了一种新工具，将有助于更深入地研究丙氨酸扩展如何影响体内丙氨酸扩展型PABPN1的聚集、定位和稳态水平，为OPMD潜在的分子机制提供新的见解。
