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J Biol Chem. 2019 Apr 30. pii: jbc.RA119.008852. doi: 10.1074/jbc.RA119.008852. [Epub ahead of print]

Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.

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Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Germany.
Institute of Biochemistry and Molecular Biology, University of Bonn, Germany.
Molecular and Integrative Biosciences Research Programme, University of Helsinki, Finland.
Leibniz-Institut fuer Molekulare Pharmakologie (FMP), Germany.
University Medical Centre Hamburg-Eppendorf.
Morphology Unit, Center for Molecular Neurobiology ZMNH, University Medical Center Hamburg-Eppendorf.
Vector Core Unit.
Center for Human Genetic Research, Massachusetts General Hospital, United States.


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 three 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 eleven 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.


electron microscopy (EM); endocytosis; lipid droplet; lipid metabolism; lysosome; mannose 6-phosphate receptors; neurodegenerative disease; pH regulation; protein sorting; proteomics; trafficking

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