title: Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C.
abstract: Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (Kd = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63-66 and 74-76, which is distinct from the region known to enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. For the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.
authors.affiliation: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892, United States. Electronic address: gruschuj@helix.nih.gov.
authors.forename: James M
authors.initials: JM
authors.lastname: Gruschus
authors.affiliation: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892, United States.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892, United States.
authors.forename: Thai Leong
authors.initials: TL
authors.lastname: Yap
authors.affiliation: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892, United States.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Hill
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Alexander
authors.initials: A
authors.lastname: Grishaev
authors.affiliation: Biophysics Core, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.affiliation: Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Ellen
authors.initials: E
authors.lastname: Sidransky
authors.affiliation: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892, United States. Electronic address: leej4@mail.nih.gov.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Lee
mesh_terms: D020134:Catalytic Domain; D005776:Gaucher Disease / Q000201:enzymology*; D005962:Glucosylceramidase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010300:Parkinson Disease / Q000201:enzymology*; D055503:Protein Multimerization; D055550:Protein Stability; D049231:Saposins / Q000737:chemistry / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism
chemical_list: D049231:Saposins; D051844:alpha-Synuclein; D005962:Glucosylceramidase
keywords: AUC; Gaucher disease; NMR; Parkinson disease; α-synuclein