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Proteomic analysis of hydrolase levels in the lysosomes from macrophages stimulated with or without LPS via liquid chromatography–mass spectrometry

ABSTRACT: Proteomic analysis of hydrolase levels in the lysosomes from macrophages stimulated with or without LPS via liquid chromatography–mass spectrometry (LC-MS), highlighting the expected LPS-induced increase in accumulation of proteases, ribonucleases (RNase) and other hydrolases.

ORGANISM(S): Mus Musculus

SUBMITTER: Wei Wei

PROVIDER: PXD056774 | iProX | Sun Oct 13 00:00:00 BST 2024

REPOSITORIES: iProX

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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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Proteomic analysis of hydrolase levels in the lysosomes from macrophages stimulated with or without LPS via liquid chromatography–mass spectrometry

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