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.
Highlights
From the ‡Department of Biochemistry, Children’s Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; §Waters Corporation, Wilmslow, SK9 4AX, United Kingdom; ¶Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands; ʈInstitut fur Biochemie, Christian-Albrechts-Universitat zu Kiel, 24098 Kiel, Germany; **Morphology Unit, Center for Molecular Neurobiology ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Soluble lysosomal proteins undergo a specific modification on high-mannose type N-linked oligosaccharides for an efficient transport from the Golgi apparatus to late endosomes and lysosomes by phosphorylation of distinct mannose residues in the C6 position. This modification is catalyzed by the subsequent action of two enzymes: the first enzyme is an N-acetylglucosamine (GlcNAc)1-1-phosphotransferase (PT), which mediates the transfer of GlcNAc-1-phosphate from uridine diphosphate (UDP)-GlcNAc to mannose acceptors forming GlcNAc-phosphomannose diester [1,2,3]
Separation of the lysosomal F2 fractions by 2D gel electrophoresis followed by Coomassie staining indicated additional differences in the composition of the lysosomal proteome between wt and previously a Gnptab knock-in (PTki) mouse liver, such as the amounts and isoform pattern for lysosomal proteases tripeptidyl peptidase 1 (Tpp1), cathepsin H (Ctsh), cathepsin Z (Ctsz), cathepsin B (Ctsb), and cathepsin D (Ctsd)
Summary
Separation of the lysosomal F2 fractions by 2D gel electrophoresis followed by Coomassie staining indicated additional differences in the composition of the lysosomal proteome between wt and PTki mouse liver, such as the amounts and isoform pattern for lysosomal proteases tripeptidyl peptidase 1 (Tpp1), cathepsin H (Ctsh), Ctsz, Ctsb, and Ctsd (supplemental Fig. S1). Abundant lysosomal membrane proteins like Lamp1, Lamp2, Limp2, and Npc1 showed no differences in their total levels in tritosomes between wt and PTki mice (Table I, supplemental Table S3).
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