Laforin, a Dual Specificity Phosphatase Involved in Lafora Disease, Is Present Mainly as Monomeric Form with Full Phosphatase Activity

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.拉弗拉病（Lafora Disease, LD）是一种致命的神经退行性癫痫性疾病，临床表现为神经功能恶化，伴随不溶性细胞内高磷酸化碳水化合物——拉弗拉小体（Lafora bodies, LBs）的积累。拉弗拉病由编码拉弗林（laforin）或马林（malin）的基因突变所致。拉弗林含有双特异性磷酸酶结构域与碳水化合物结合模块，属于新近被报道的葡聚糖磷酸酶家族成员。在本研究中，我们探究了拉弗林二聚化的功能与生理学相关性。我们纯化了重组人源拉弗林，并将其单体与二聚体组分分别进行变性凝胶电泳、质谱分析、磷酸酶活性测定、蛋白-蛋白相互作用实验以及葡聚糖结合实验。实验结果表明，无论在体外还是体内环境中，拉弗林均主要以单体形式存在，仅伴随少量二聚体组分。从机制层面来看，拉弗林单体与二聚体具有同等的磷酸酶活性，二者均能与马林蛋白结合，且与葡聚糖的结合程度相似。但我们发现，两种聚合状态在同时与马林蛋白和碳水化合物相互作用的能力上存在差异。此外，我们还对葡聚糖磷酸酶家族的其他成员进行了测试。综合所有实验数据，我们的研究结果表明，拉弗林单体是该蛋白的主要存在形式，且具备磷酸酶活性。