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Abstract
Saposins are helix bundle proteins which solubilize sphingolipids and present them to lysosomal hydrolases for catabolism. Saposin B (SapB) is an activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A (GLA). The mechanism by which SapB activates GLA is unknown. SapB forms dimeric water-soluble lipoprotein complexes in vitro and presents a restrictive conformation in crystal structures. To uncover the molecular mechanism of SapB presenting to GLA, we subjected the fluorescent substrate derivate Gb3NBD to a series of assays involving SapB. Firstly, we showed that SapB stably binds Gb3NBD and presents it to GLA for cleavage in vitro. Secondly, we crystallized SapB in the presence of Gb3NBD. Thirdly, we showed that transient interactions between SapB and GLA can be chemically cross-linked. Fourthly, we crystallized SapB in the presence of detergent, which detergent also led to the capture of a binary complex between SapB and GLA. These findings provide direction for future biochemical and structural studies on the remaining saposins and their cognate hydrolases.
Type
dissertation
Date
2022-05
Publisher
Degree
Advisors
License
License
http://creativecommons.org/licenses/by/4.0/