Determining the Role of Lafora Disease Patient Mutations on Glycogen Dephosphorylation.

Abstract

Lafora Disease (LD) is fatal, congenital neurodegenerative epilepsy caused by mutations in the gene that encodes the protein laforin. These mutations disrupt glycogen metabolism in cells causing accumulations of malformed glycogen inclusions called Lafora Bodies (LB). LBs inhibit normal neuronal functions, leading to epilepsy, dementia, and death by age 25. Laforin contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. To date, more than 40 laforin point mutations have been identified in LD patients. However, how each of these mutations leads to LB formation and/or how they impact glycogen dephosphorylation is not known. Our lab recently determined the 3‐dimensional structure of laforin using X‐ray crystallography. The laforin X‐ray crystal structure reveals that these patient point mutations localize in four different regions within the DSP and CBM domains suggesting multiple possible disease mechanisms. To determine the role of each of these mutations on glycogen dephosphorylation, we employed a generic pNPP phosphatase activity assay and a biologically relevant assay utilizing glycogen to measure glycogen phosphatase activity. The glycogen phosphatase assay was very specific and faithfully reported both wildtype and mutant laforin phosphatase activity. Mutations that are located in the CBM‐DSP interface and the DSP‐DSP dimer interface resulted in 50‐80% decrease, suggesting the importance of these regions in optimal laforin activity. We found a 70‐90% decrease in glycogen phosphatase activity for mutations that are localized in the CBM binding site and the DSP active site.