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

Molecular & Cellular Proteomics : MCP
Giorgia Di LorenzoSandra Pohl

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 s...Continue Reading

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Citations

Aug 4, 2018·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Sandra PohlThorsten Schinke
Jul 10, 2020·Journal of the American Society of Nephrology : JASN·Wiebke SachsCatherine Meyer-Schwesinger
Aug 4, 2019·International Journal of Molecular Sciences·Xiaoai FangXia Cai
Jan 19, 2021·EMBO Molecular Medicine·Giancarlo ParentiAndrea Ballabio
Aug 4, 2021·Genetics in Medicine : Official Journal of the American College of Medical Genetics·Giorgia Di LorenzoSandra Pohl
Sep 4, 2021·Molecular Omics·Pathma Muthukottiappan, Dominic Winter

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