title: Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.
abstract: The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver.
authors.affiliation: From the ‡Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.|§Waters Corporation, Wilmslow, SK9 4AX, United Kingdom.
authors.forename: Sandra
authors.initials: S
authors.lastname: Markmann
authors.affiliation: From the ‡Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.|§Waters Corporation, Wilmslow, SK9 4AX, United Kingdom.
authors.forename: Svenja
authors.initials: S
authors.lastname: Krambeck
authors.affiliation: §Waters Corporation, Wilmslow, SK9 4AX, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hughes
authors.affiliation: ¶Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands.
authors.forename: Mina
authors.initials: M
authors.lastname: Mirzaian
authors.affiliation: ¶Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
authors.affiliation: ‖Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany.
authors.forename: Paul
authors.identifier: 0000-0003-2637-7052
authors.initials: P
authors.lastname: Saftig
authors.affiliation: **Morphology Unit, Center for Molecular Neurobiology ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Schweizer
authors.affiliation: §Waters Corporation, Wilmslow, SK9 4AX, United Kingdom.
authors.forename: Johannes P C
authors.initials: JP
authors.lastname: Vissers
authors.affiliation: From the ‡Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; braulke@uke.de mdamme@biochem.uni-kiel.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.affiliation: ‖Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany; braulke@uke.de mdamme@biochem.uni-kiel.de.
authors.forename: Markus
authors.identifier: 0000-0002-9699-9351
authors.initials: M
authors.lastname: Damme
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D006867:Hydrolases / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D009081:Mucolipidoses / Q000201:enzymology* / Q000235:genetics; D010770:Phosphotransferases / Q000172:deficiency; D020543:Proteome / Q000032:analysis*
chemical_list: D008360:Mannosephosphates; D020543:Proteome; C027693:mannose-6-phosphate; D010770:Phosphotransferases; D006867:Hydrolases