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Transcriptional control of lysosomal homeostasis by the disease-related CLN3 membrane protein

ABSTRACT: CLN3 is a type II transmembrane protein localized in the late endosomal/lysosomal compartment. A deficiency of CLN3 leads to the development of a certain type of Neuronal Ceroid Lipofuscinosis, a neurodegenerative disorder of childhood caused by aggregation of undegraded material in the lysosomal compartment. Cultured, immortalized wild type and Cln3(Δex7/8) cerebellar granule cells were used in this project to determine the influence of Cln3 deficiency on the proteome of the lysosomal compartment. For this purpose, cells were labelled by stable amino acid labelling in cell culture and lysosomes were isolated by magnetic beads.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cerebellar Granule Cell, Cell Culture

DISEASE(S): Lysosomal Storage Disease

SUBMITTER: Marc Sylvester

LAB HEAD: Thomas Braulke

PROVIDER: PXD004548 | Pride | 2019-05-13

REPOSITORIES: Pride

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Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.

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Transcriptional control of lysosomal homeostasis by the disease-related CLN3 membrane protein

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