Abstract
Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.
Highlights
Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids
We performed SILAC-based comparative proteomics and quantified the relative amounts of lysosomal proteins at steady state in lysosomal fractions isolated from WT and Cln3⌬ex7/8 cerebellar cells (PXD004548; lysosomal proteome analysis) exhibiting storage material (Fig. S1) by means of internalized dextran-stabilized magnetite transported to LysoTracker-positive organelles (Fig. S2)
The homeostasis of lysosomes relies on the balance between the biosynthetic replenishment of lysosomal components, degradation, and recycling of macromolecules obtained by endocytosis and autophagy
Summary
Lysosomal proteomics identifies differential abundance of acid hydrolases in Cln3⌬ex7/8 cerebellar cells. In agreement with the comparative proteomic data, the activities of Ppt and Gla, representing the most enriched proteins in lysosomal fractions of Cln3⌬ex7/8 cells, were significantly increased, whereas the activities of Hexb, Arsa, Manba, and Gusb were reduced in Cln3⌬ex7/8 cells (Fig. 1C). The proteomic analysis of lysosomal fractions revealed a 2.2fold increased concentration of Mpr300 in Cln3⌬ex7/8 cerebellar cells compared with WT controls (Fig. 3A) which was confirmed by Western blotting (Fig. 3B). The comparative lipidome analyses of cerebellar cells revealed distinct alterations, in particular in the relative contents of glycosphingolipids in Cln3⌬ex7/8 cells, which can be partially explained by changes in the steadystate expression levels of lysosomal sphingolipid-degrading hydrolases. The amount of Trf-Alexa Fluor 546 detected intracellularly after 30-min incubation was significantly increased in Cln3⌬ex7/8 cerebellar cells compared with WT cells (Fig. 6, A and D). These data demonstrate that the exocytic pathway of Trf from the recycling endosome to the plasma membrane is strongly disturbed in Cln3⌬ex7/8 cells
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