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Role of the Γ-Subunit of Glcnac-1-Phosphotransferase in the Pathogenesis of Mucolipidosis Type III

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Role of the Γ-Subunit of Glcnac-1-Phosphotransferase in the Pathogenesis of Mucolipidosis Type III Dissertation with the aim of achieving the degree doctor rerum naturalium Role of the γ-subunit of GlcNAc-1-phosphotransferase in the pathogenesis of mucolipidosis type III submitted by Giorgia Di Lorenzo from Giulianova (TE) Italy at the Department of Biology, Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg Hamburg, 2018 To my family Academic advisor: Prof. Dr. Thomas Braulke Co-advisors: Prof. Dr. Tim-Wolf Gilberger Date of disputation: 22nd June 2018 Index Index 1. Introduction ........................................................................................... 1 1.1 Synthesis and transport of lysosomal protein .................................................................. 1 1.1.1 Biosynthesis of soluble lysosomal proteins ...................................................................... 1 1.1.2 Generation of mannose 6-phosphate residues on lysosomal enzymes .............................. 2 1.1.3 GlcNAc-1-phosphotransferase .......................................................................................... 3 1.2 Mucolipidosis type II and III ............................................................................................ 5 1.3 Bone formation and remodelling ...................................................................................... 8 1.4 Skeletal abnormalities in MLII mice .............................................................................. 10 1.5 Animal models for MLIII ................................................................................................ 11 2. Aim of the study ................................................................................... 14 3. Materials and Methods ....................................................................... 15 3.1 Materials ........................................................................................................................... 15 3.1.1 Chemicals ........................................................................................................................ 15 3.1.2 Equipment ....................................................................................................................... 16 3.1.3 Consumables ................................................................................................................... 17 3.1.4 Kits and assay .................................................................................................................. 17 3.1.5 Enzymes .......................................................................................................................... 17 3.1.6 Enzyme substrates ........................................................................................................... 18 3.1.7 Primera and TaqMan™ assays ........................................................................................ 18 3.1.8 Media and supplements for cell culture .......................................................................... 19 3.1.9 Antibodies ....................................................................................................................... 20 3.1.10 Software ........................................................................................................................ 21 3.2 Molecular biology methods ............................................................................................. 21 3.2.1 Genotyping of mice ......................................................................................................... 21 3.2.2 Agarose gel electrophoresis ............................................................................................ 22 3.2.3 RNA extraction and cDNA synthesis.............................................................................. 22 3.2.4 Quantitative real-time PCR ............................................................................................. 22 3.3 Cell biology methods ........................................................................................................ 23 3.3.1 Isolation and culture of mouse embryonic fibroblasts .................................................... 23 3.3.2 Cryo-conservation and revitalization of MEF ................................................................. 24 Index 3.3.3 Isolation of bone marrow cells for osteoclasts and osteoblasts cultivation ..................... 24 3.3.4 Isolation and culture of calvarial osteoblasts .................................................................. 25 3.3.5 Isolation and culture of ribcage chondrocytes ................................................................. 25 3.3.6 Alizarin red staining ........................................................................................................ 26 3.3.7 Preparation of conditioned media ................................................................................... 26 3.3.8 Stable isotope labelling by amino acids in cell culture (SILAC) .................................... 26 35 3.3.9 Metabolic SO4-labeling of MEF and chondrocytes ...................................................... 26 3.4 Biochemical methods ....................................................................................................... 27 3.4.1 Preparation of cell extracts d culture of mouse embryonic fibroblasts ........................... 27 3.4.2 Media preparation ........................................................................................................... 27 3.4.3 Sample preparation for mass spectrometry ..................................................................... 27 3.4.4 Preparation of lysosomal-enriched fractions ................................................................... 28 3.4.5 Protein quantification ...................................................................................................... 28 3.4.6 SDS polyacrylamide gel electrophoresis (SDS-PAGE) .................................................. 28 3.4.7 Western blot analysis ...................................................................................................... 29 3.4.8 Enzyme activity measurements ....................................................................................... 29 3.4.9 Purification of GAGs by anion exchange chromatography ............................................ 31 3.5 Histochemical methods .................................................................................................... 32 3.5.1 Examinations of Gnptg expression by Lacz staining ...................................................... 32 3.5.2 Histological examinations on tissue sections by electron microscopy ............................ 33 3.5.3 Histomorphometric analysis and µCT of bone tissue ..................................................... 33 3.6 Statistical analysis ............................................................................................................ 33 4. Results ................................................................................................... 34 4.1 Expression analysis of GlcNAc-1-phosphotransferase ................................................. 34 4.2 Role of the γ-subunits for M6P formation on lysosomal enzymes ............................... 37 4.2.1 Molecular and biochemical analysis of Gnptgko mice ..................................................... 37 4.2.2 Role of the γ-subunits for GlcNAc-1-phosphotransferase activity ................................. 38 4.2.3 Lysosomal proteome of Gnptgko MEF ............................................................................ 40 4.2.4 Validation of the lysosomal proteome analysis ............................................................... 43 4.2.5 M6P proteome of Gnptgko MEF ...................................................................................... 45 4.2.6 Loss of Arsb causes accumulation of GAGs in Gnptgko MEF ........................................ 47 4.3 Role -subunits for bone and cartilage homeostasis ...................................................... 51 4.3.1 Bone architecture of Gnptgko mice .................................................................................. 51 4.3.2 Analysis of storage material in bone and cartilage in Gnptgko mice ............................... 54 Index 4.3.3 Differentiation and mineralization of Gnptgko osteoblasts .............................................. 55 4.3.4 Trafficking of lysosomal enzymes in osteoblasts and osteocytes ................................... 56 4.3.5 Analysis of storage material in cartilage in Gnptgko mice ............................................... 57 4.3.6 Loss of Arsb causes accumulation of GAGs in Gnptgko chondrocytes ........................... 58 5. Discussion ............................................................................................. 60 5.1 Reduced GlcNAc-1-phosphotransferase activity in the absence of γ-subunits ........... 60 5.2 The role of γ-subunits for bone and cartilage homeostasis........................................... 64 5.3 Enzyme replacement for reduction of GAG accumulation in Gnptgko cells ............. 68 6. Summary .............................................................................................. 74 7. References ............................................................................................ 75 8. Publications, conference contributions and awards ........................ 84 8.1 Publications ......................................................................................................................
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