Dimerization of the Glucan Phosphatase Laforin Requires the Participation of Cysteine 329

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.

拉福林蛋白（Laforin）由与拉福拉病（Lafora Disease, LD, OMIM 254780）相关的突变基因编码，是一种由碳水化合物结合模块（carbohydrate-binding module）与双特异性磷酸酶结构域（dual-specificity phosphatase domain）构成的模块化蛋白。拉福林蛋白是葡聚糖磷酸酶家族的首个成员，可调控糖原中的磷酸根水平。多项研究已证实拉福林蛋白可形成二聚体，但此类二聚体的功能及其与拉福拉病的关联仍未明确。近期研究证据表明，拉福林蛋白的二聚化过程依赖于氧化还原环境，提示二硫键参与了其二聚化过程。本研究通过定点诱变（site-directed mutagenesis）技术构建了一系列拉福林蛋白突变体，将其中的单个半胱氨酸残基替换为丝氨酸；随后分别通过重组蛋白（recombinant protein）实验与哺乳动物细胞培养实验（mammalian cell culture assay），检测各突变体的二聚化能力。仅拉福林-Cys329Ser（Laforin-Cys329Ser）突变体在两类实验中均无法形成二聚体。本研究还构建了缺失最后三个氨基酸的拉福林蛋白截短体laforin-Cys329X，该截短体同样无法发生二聚化。值得注意的是，laforin-Cys329Ser与laforin-Cys329X仍可结合葡聚糖，且对外源底物及生理相关底物均保留了野生型磷酸酶活性。此外，laforin-Cys329Ser可完全参与由拉福林-马林复合物（laforin-malin complex）介导的泛素化（ubiquitination）过程。上述结果表明，二聚化并非拉福林蛋白发挥磷酸酶活性、结合葡聚糖，或是形成功能性拉福林-马林复合物所必需的过程。综合来看，上述结果提示第329位半胱氨酸特异性参与了拉福林蛋白的二聚化过程。因此，C329S突变体可作为研究拉福林蛋白寡聚化生理意义的有效工具。