biostudies-literatureUnknownSawyer TKNIDDK NIH HHSNIGMS NIH HHSSphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates α-galactosidase A cleavage of Gb3-NBD <i>in vitro</i>. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactiobioRxiv : the preprint server for biology2024.04.04.584535https://www.ebi.ac.uk/biostudies/studies/S-EPMC11014568biostudies-literatureHuman Saposin B Ligand Binding and Presentation to α-Galactosidase A.PMC11014568P30 GM133894R01 DK076877Garman SCStaros JVBobst CEAral ESawyer TKfalseHuman Saposin B Ligand Binding and Presentation to α-Galactosidase A.Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates α-galactosidase A cleavage of Gb3-NBD <i>in vitro</i>. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactio2024-01-01T00:00:00Z2024 Apr2026-05-27T08:04:10.047Z2026-05-27T03:07:56.564ZS-EPMC110145683861723610.1101/2024.04.04.584535