Lafora病(LD)是一种常染色体隐性遗传性肌阵挛性癫痫,在青少年时期发作,导致发病十年内死亡。LD的特征是过度积累的过度磷酸化,分枝差,不溶性,糖原样聚合物称为Lafora体。这种疾病是由EPM2A的突变引起的,编码拉福林,一种去磷酸化糖原的双特异性磷酸酶,或EMP2B,编码马林,E3-泛素连接酶。虽然糖原是一种被广泛接受的laforin底物,malin的底物难以鉴定,部分原因是缺乏能够在体内检测到malin的malin抗体。在这里,我们描述了一个小鼠模型,其中malin基因在C端被修饰以包含c-myc标签序列,使malin-myc的表达易于检测。使用c-myc标签抗体对免疫沉淀物进行的质谱分析表明,Malin与Laforin和许多糖原代谢酶相互作用。为了研究laforin在这些相互作用中的作用,我们分析了另外两种小鼠模型:malin-myc/laforin敲除和malin-myc/LaforinCS,其中laforin不存在或催化Cys基因组突变为Ser,分别。Malin与伴侣蛋白的相互作用需要Laforin,但不依赖于其催化活性或糖原的存在。总的来说,结果表明,拉福林和马林在体内形成复合物,它能稳定苹果酸并增强与伴侣蛋白的相互作用,以促进正常的糖原代谢。他们还提供了有关LD发展和通过催化失活磷酸酶挽救疾病的见解。
Lafora disease (LD) is an autosomal recessive myoclonus epilepsy with onset in the teenage years leading to death within a decade of onset. LD is characterized by the overaccumulation of hyperphosphorylated, poorly branched, insoluble, glycogen-like polymers called Lafora bodies. The disease is caused by mutations in either EPM2A, encoding
laforin, a dual specificity phosphatase that dephosphorylates glycogen, or EMP2B, encoding malin, an E3-ubiquitin ligase. While glycogen is a widely accepted
laforin substrate, substrates for malin have been difficult to identify partly due to the lack of malin antibodies able to detect malin in vivo. Here we describe a mouse model in which the malin gene is modified at the C-terminus to contain the c-myc tag sequence, making an expression of malin-myc readily detectable. Mass spectrometry analyses of immunoprecipitates using c-myc tag antibodies demonstrate that malin interacts with laforin and several glycogen-metabolizing enzymes. To investigate the role of laforin in these interactions we analyzed two additional mouse models: malin-myc/
laforin knockout and malin-myc/LaforinCS, where
laforin was either absent or the catalytic Cys was genomically mutated to Ser, respectively. The interaction of malin with partner proteins requires
laforin but is not dependent on its catalytic activity or the presence of glycogen. Overall, the results demonstrate that
laforin and malin form a complex in vivo, which stabilizes malin and enhances interaction with partner proteins to facilitate normal glycogen metabolism. They also provide insights into the development of LD and the rescue of the disease by the catalytically inactive phosphatase.