Metabolism of Glycosphingolipids and targeting GM2 synthesis pathway to develop substrate reduction approach in Tay-Sachs and Sandhoff disorders.

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Abidi, Iram
Lysosomal Storage Disorders, Glycosphingolipids
GM2 gangliosidosis is a rare genetic lysosomal storage disorder (LSD) in children, with no effective therapies available presently. Tay-Sachs (TSD) and Sandhoff disorders (SD) are caused by a disruption of the catabolic pathway of gangliosides in lysosomes, accumulating GM2 and lyso-GM2, which damage cells and tissues. This leads to symptoms that often include neurological deterioration, such as cognitive decline, motor dysfunction, and seizures. For the purpose to develop substrate reduction therapy (SRT) for GM2 gangliosidosis in TSD and SD, we studied the enzyme beta1,4-N-acetylgalactosaminyltransferase 1, B4GALNT1, responsible for the synthesis of GM2. We attempted to predict its structural features by modelling the enzyme using Bioinformatics tools and characterized B4GALNT1 activity to establish its properties, stability, and inhibition. Additionally, we established an enzyme assay to produce Lyso-GM2 from GM2 using an in-vitro method, to utilise Lyso-GM2 as a possible biomarker for detection of TSD and SD. We used eukaryotic transient expression systems (HEK293 and Expi293cell lines) to express B4GALNT1 in vitro. Western blots demonstrated production of soluble protein in Expi293, which was successfully purified using Ni-NTA chromatography. The predicted 3D structure of B4GALNT1 established highly conserved residues, showing a potential catalytic DXD motif at position 356-358, close to residues Y501 and H483 which are likely to be important for donor binding. R505 is another significant amino acid reported to be mutated in a small number of patients with GM2 gangliosidosis. B4GALNT1 was strongly inhibited by bis-imidazolium salts that are selective inhibitors of glycosyltransferases. These inhibitors could decrease the synthesis of GM2 and other related glycosphingolipids in the biosynthetic pathway, and further avoid accumulation of GM2 and lyso-GM2 in patients with patients with lysosomal dysfunctionalities and neurodegeneration like TSD and SD. This work can lead to potential therapies for these disorders.
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