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Author ORCID Identifier
https://orcid.org/0000-0002-3600-210X
AccessType
Open Access Dissertation
Document Type
dissertation
Degree Name
Doctor of Philosophy (PhD)
Degree Program
Molecular and Cellular Biology
Year Degree Awarded
2022
Month Degree Awarded
May
First Advisor
Scott C. Garman
Subject Categories
Biochemistry, Biophysics, and Structural Biology
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.
DOI
https://doi.org/10.7275/28612792
Recommended Citation
Sawyer, Thomas K., "Human Saposin B Ligand Binding and Presentation to α-Galactosidase A" (2022). Doctoral Dissertations. 2569.
https://doi.org/10.7275/28612792
https://scholarworks.umass.edu/dissertations_2/2569
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.