UC San Diego

Lafora Disease (LD) is a fatal neurodegenerative disease that is correlated with mutation of the human phosphatase, Laforin. While Laforin has been shown to dephosphorylate phosphoglucans, little is known about the mechanism of dephosphorylation and Laforin's structure. Laforin consists of two domains: a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. The absence of Laforin function results in hyperphosphorylated and poorly branched sugar accumulations. It is hypothesized that these insoluble sugar deposits lead to neurodegeneration and pre-mature death in LD patients. We utilized hydrogen/deuterium exchange mass spectrometry (DXMS) to define Laforin's structural components and to probe Laforin's substrate interactions. DXMS analysis was performed on wild-type (WT) Laforin and LD mutants : W32G, G240S, and Y294N. The analysis of WT Laforin revealed the strongest substrate interaction with glycogen, when compared to interactions with the carbohydrates amylopectin and [Beta]-cyclodextrin. WT data revealed that regions of the CBM were protected from deuteration when bound to glucans. Similarly, structurally important regions of the DSP showed deuteration protection. W32G data confirmed the lack of protection from deuteration in the absence of substrate binding, whereas G240S data confirmed deuteration protection due to substrate binding. Lastly, the Y294N data revealed surprisingly strong substrate interactions in the DSP mutation region, despite weak CBM interaction. Our results confirm the role of Laforin's CBM in glucan binding and highlight the significance of structural DSP elements in the dephosphorylation of phosphoglucans