Structural Characterization of Saposin D Binding Properties for Determining the Molecular Basis of Farber Disease
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Tharp, Claire Dorothy
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Abstract
Farber disease is a rare lysosomal storage disorder caused by impaired degradation of the ceramide sphingolipid due to mutations in the lysosomal hydrolase enzyme acid ceramidase (ACDase) and the lipid protein transfer saposin D. While saposin D is known to enhance ACDase activity, the molecular mechanism underlying formation of the complex and transfer of lipids between the two is unclear. Here, solution NMR spectroscopy was used to characterize the structure and binding properties of saposin D under its native lysosomal conditions. The NMR structure of saposin D at pH 4.0 revealed a stable, monomeric four-helix bundle in a closed conformation. NMR titration experiments demonstrated that saposin D binds multiple lipid classes through a conserved surface. In particular, a localized patch of conserved cationic Lys and Arg residues on saposin D were identified to interact with anionic lipid headgroups. Further, the same saposin D surface was found to be involved in interaction with ACDase, with significant overlap in the residues involved in lipid and enzyme binding. These findings support a model of interaction in which saposin D utilizes a charged surface to extract anionic lipids from cellular membranes and present them to ACDase, facilitating ceramide hydrolysis. This work provides a structural framework for understanding the role of saposin D and ACdase in sphingolipid metabolism and offers insight into how genetic mutations contribute to Farber disease.
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2026-05
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Undergraduate Thesis