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Mol Cell Proteomics. 2018 Aug;17(8):1612-1626. doi: 10.1074/mcp.RA118.000720. Epub 2018 May 17.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Author information

1
From the ‡Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
§Institute of Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany.
3
¶Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
4
‖Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
5
**Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
6
‡‡Division Biophysics, Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany.
7
From the ‡Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; s.pohl@uke.de braulke@uke.de.

Abstract

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2β2γ2). Upon proteolytic cleavage by site-1 protease, the α/β-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/β-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

KEYWORDS:

Affinity proteomics; Animal models; Enzyme modification; Gnptg-deficient mouse; Post-translational modifications; SILAC; arylsulfatase B; lysosomal missorting; lysosomal proteome; mannose 6-phospho-secretome

PMID:
29773673
PMCID:
PMC6072544
[Available on 2019-08-01]
DOI:
10.1074/mcp.RA118.000720

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