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Structure of human saposin A at lysosomal pH.

ABSTRACT: The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme ?-galactocerebrosidase (GALC), which catalyzes the breakdown of ?-D-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a `closed' to an `open' conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8?Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined `closed' conformation, showing that pH alone is not sufficient for the transition to the `open' conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.

SUBMITTER: Hill CH

PROVIDER: S-EPMC4498711 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Structure of human saposin A at lysosomal pH.

Hill Chris H CH Read Randy J RJ Deane Janet E JE

Acta crystallographica. Section F, Structural biology communications 20150627 Pt 7

The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-D-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from ...[more]

PMID: 26144235

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Project description:Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and alpha-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using alpha-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of alpha-Gal A with DGJ. Both GCase and alpha-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in alpha-Gal A are not seen. Thermodynamic parameters obtained from alpha-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and alpha-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.

Dataset Information

Structure of human saposin A at lysosomal pH.

Publications

Structure of human saposin A at lysosomal pH.

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