Bueno Carlos, Tabares-Seisdedos Rafael, Moraleda Jose M, Martinez Salvador
IMIB-Arrixaca and Faculty of Medicine, University of Murcia, Murcia and CIBERSAM, Murcia, Spain.
Teaching Unit of Psychiatry and Psychological Medicine, Department of Medicine, University of Valencia and CIBERSAM, Valencia, Spain.
PLoS One. 2016 Apr 11;11(4):e0153262. doi: 10.1371/journal.pone.0153262. eCollection 2016.
Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may exist in multiple different molecular forms and that molecular pattern variations derived from altered post-transcriptional processing may underlay Rett syndrome physiophatology.
MeCP2蛋白功能障碍会导致多种神经疾病，如雷特综合征和自闭症。MeCP2蛋白的确切功能仍远未明确。在分子水平上，存在相互矛盾的数据。通过蛋白质免疫印迹分析，MeCP2蛋白被认为是一条约75 kDa的单一免疫反应条带，但有几份报告显示，在预期的MeCP2水平之上和之下存在多条MeCP2免疫反应条带。对MeCP2免疫反应条带的解释各不相同。一些研究人员认为，多条MeCP2免疫反应条带是与MeCP2抗体发生交叉反应的未鉴定蛋白质或MeCP2的降解产物，而另一些人则认为，MeCP2的转录后加工产生了与细胞信号传导相关的多种分子形式，但到目前为止，它们尚未在雷特综合征实验模型中得到恰当分析。本研究的目的是加深对对照神经细胞和p.T158M MeCP2e1突变细胞中多条MeCP2免疫反应条带的理解。我们构建了稳定表达野生型和p.T158M MeCP2e1-RFP突变体的细胞。应用N端和C端MeCP2抗体以及RFP抗体，最大限度地减少了对非特异性交叉反应的担忧，因为它们在不同表位与同一抗原发生反应。我们报告了在对照细胞、稳定表达野生型和p.T158M MeCP2e1-RFP突变体的细胞中存在多条MeCP2免疫反应条带。此外，在野生型和表达p.T158M MeCP2e1-RFP突变体的细胞之间发现了MeCP2免疫反应条带的差异。在表达p.T158M MeCP2e1-RFP突变体的细胞中，约70kDa的迁移较慢的磷酸化条带消失。这些数据表明，苏氨酸158可能代表一个潜在参与蛋白质功能的重要磷酸化位点。我们的结果清楚地表明，MeCP2抗体与其他蛋白质上的相似表位没有交叉反应，支持了MeCP2可能以多种不同分子形式存在的观点，并且转录后加工改变导致的分子模式变化可能是雷特综合征病理生理学的基础。
