Instituto de Biomedicina de Valencia, CSIC and Centro de Investigación Biomédica en Red de Enfermedades Raras, Valencia, Spain.
PLoS One. 2013 Jul 26;8(7):e69523. doi: 10.1371/journal.pone.0069523. Print 2013.
Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin's oligomerization.
拉弗林由一个在拉佛拉病（LD，OMIM 254780）中发生突变的基因编码，是一种由碳水化合物结合模块和双特异性磷酸酶结构域组成的模块化蛋白质。拉弗林是糖基磷酸酶家族的创始成员，调节糖原中存在的磷酸水平。多项报道描述了拉弗林形成二聚体的能力，尽管这些二聚体的功能及其与 LD 的关系仍不清楚。最近的证据表明，拉弗林二聚化依赖于氧化还原条件，表明二硫键参与拉弗林二聚化。我们使用定点突变构建了单个半胱氨酸残基被丝氨酸取代的拉弗林突变体，然后使用重组蛋白和哺乳动物细胞培养测定来测试每种蛋白质形成二聚体的能力。拉弗林-Cys329Ser 是唯一一种在两种测定中都不能形成二聚体的 Cys/Ser 突变体。我们还生成了一个缺失最后三个氨基酸的拉弗林截短体，拉弗林-Cys329X，该截短体也不能二聚化。有趣的是，拉弗林-Cys329Ser 和拉弗林-Cys329X 能够结合葡聚糖，并保持对外源性和生物学相关底物的野生型磷酸酶活性。此外，拉弗林-Cys329Ser 完全能够参与由拉弗林-马拉林复合物驱动的泛素化过程。这些结果表明，二聚化不是拉弗林磷酸酶活性、葡聚糖结合或功能性拉弗林-马拉林复合物形成所必需的。总之，这些结果表明半胱氨酸 329 特异性参与拉弗林的二聚化过程。因此，C329S 突变体构成了分析拉弗林寡聚化生理影响的有价值工具。
