Aus der Klinik und Poliklinik im Dr. von Haunerschen Kinderspital Klinik der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Christoph Klein Probe the transport function of ABCA3 by metabolic labelling of choline phospholipids Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Yang Li aus Sichuan, China 2020 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. med. Matthias Griese Mitberichterstatter: Prof. Dr. med. Erika von Mutius Prof. Dr. med. Albrecht Bergner Mitbetreuung durch den promovierten Mitarbeiter: Dekan: Prof. Dr. med. dent: Reinhard Hickel Tag der mündlichen Prüfung: 17.09.2020 Content ABSTRACT .............................................................................................................................. 1 1 INTRODUCTION ............................................................................................................ 2 1.1 PULMONARY SURFACTANT .............................................................................................. 2 1.1.1 Synthesis of surfactant lipids .................................................................................. 2 1.1.2 Composition of surfactant ...................................................................................... 4 1.1.3 Transport of surfactant between sub organelles .................................................... 5 1.1.4 Lamellar bodies ...................................................................................................... 6 1.1.5 Function of surfactant lipids .................................................................................. 7 1.2 ABCA3 ........................................................................................................................... 7 1.2.1 ABC transporter family .......................................................................................... 7 1.2.2 Biogenesis and structure of ABCA3 ....................................................................... 8 1.2.3 Function of ABCA3 .............................................................................................. 11 1.2.4 ABCA3 mutations and related pulmonary diseases ............................................. 11 1.2.5 Treatment of ABCA3 mutations related pulmonary disease ................................ 13 1.2.6 Functional assay of ABCA3 ................................................................................. 14 2 AIMS ............................................................................................................................... 17 3 ZUSAMMENFASSUNG ............................................................................................... 18 4 MATERIAL AND METHODS ..................................................................................... 19 4.1 MATERIALS ................................................................................................................... 19 4.2 METHODS ...................................................................................................................... 27 4.2.1 Site directed mutagenesis ..................................................................................... 27 4.2.2 Plasmid preparation ............................................................................................. 28 4.2.3 Agarose gel electrophoresis ................................................................................. 28 4.2.4 Sequencing ........................................................................................................... 28 4.2.5 Transfection and single clone generation ............................................................ 29 4.2.6 Cell culture ........................................................................................................... 30 4.2.7 BCA assay ............................................................................................................ 30 4.2.8 Western blot .......................................................................................................... 30 4.2.9 cDNA synthesis ..................................................................................................... 31 4.2.10 Quantitative polymerase chain reaction .............................................................. 32 4.2.11 Propargyl-choline treatment ................................................................................ 32 4.2.12 Inhibitors .............................................................................................................. 33 4.2.13 Immunofluorescent staining ................................................................................. 33 4.2.14 Click reaction ....................................................................................................... 33 4.2.15 Imaging ................................................................................................................. 34 4.2.16 Fluorescence intensity analyzing ......................................................................... 34 4.2.17 Cytotoxicity assay ................................................................................................. 35 4.2.18 Lipids Mass-spectrometry .................................................................................... 36 4.2.19 Cell homogenization ............................................................................................. 38 4.2.20 Mark lamellar bodies with Lysotracker Red ........................................................ 38 4.2.21 Lamellar body isolation ....................................................................................... 39 4.2.22 Statistical analysis ................................................................................................ 39 5 RESULTS ........................................................................................................................ 40 5.1 ESTABLISH AN ASSAY TO PROBE THE PHOSPHATIDYL-CHOLINE TRANSPORT FUNCTION OF ABCA3 ................................................................................................................................. 40 5.1.1 Construction of ABCA3-HA stable expressing cells ............................................ 40 5.1.2 Incorporation of propargyl-Cho into phospholipids ............................................ 42 5.1.3 Incorporation of propargyl-Cho in an ATP dependent manner .......................... 46 5.1.4 Incorporation of propargyl-Cho was concentration dependent .......................... 47 5.1.5 Incorporation of propargyl-Cho was time dependent .......................................... 47 5.1.6 Competitive inhibition phosphatidyl-propargyl-Cho transport ........................... 50 5.1.7 Irreversibly inhibition of phosphocholine synthesis by MN58b ........................... 51 5.1.8 Deviation of transport function of p.N568D and p.L1580P ABCA3 mutation .... 53 5.2 LAMELLAR BODY ISOLATION ......................................................................................... 55 5.2.1 Separation lamellar bodies by sucrose gradient .................................................. 55 5.2.2 Intactness of fractionated lamellar bodies ........................................................... 55 5.2.3 Purity of fractionated lamellar bodies ................................................................. 56 6 DISCUSSION ................................................................................................................. 58 7 PUBLICATION .............................................................................................................. 68 8 AFFIDAVIT/ EIDESSTATTLICHE VERSICHERUNG .......................................... 69 9 ACKNOWLEDGEMENTS ........................................................................................... 70 10 REFERENCE ................................................................................................................. 71 Index of Figures Figure 1: Alveolar epithelial cells………………………………………………………2 Figure 2: De novo pathway. ……………………………………………………………3 Figure 3: Lipids and protein composition of pulmonary surfactant. …….…………5 Figure 4: Biogenesis and 3D structure of ABCA3. …………...………………………9 Figure 5: Ultra-structure of ABCA3. ………………………….………………………10 Figure 6: Propargyl-choline and click reaction of labeled PC. ………………………15 Figure 7: Region of interest (ROI). ….…………………………………………………35 Figure 8: ABCA3-HA stable expressing A549 cells. ……………….…………………40 Figure 9: Mean peak areas of propargyl-Cho labeled lipids. ……………………………42 Figure 10: Mean peak areas of choline head lipids. ………………………………………43 Figure 11: Transfected A549 cells successfully incorporated propargyl-Cho.……...……44 Figure 12: Distribution of propargyl-Cho labeled lipids. …………………………….…45 Figure 13: Toxicity of propargyl-Cho. ….………………………………………….……46 Figure 14: Incorporation of propargyl-Cho in an ATP dependent manner……………46 Figure 15: Incorporation of propargyl-Cho was concentration dependent. ………...…48 Figure 16: Incorporation of propargyl-Cho was time dependent. ……………….……49 Figure 17: Competitive inhibit phosphatidyl-propargyl-Cho transportation of ABCA3...50 Figure 18: Irreversibly inhibit the phosphocholine synthesis by MN58b…………………52 Figure 19: Deviation of transport function of p.N568D and p.L1580P ABCA3 mutation...53 Figure 20: Isolation of lamellar bodies by sucrose gradient……………………………….55 Figure 21: Intactness of fractionated lamellar bodies…………….………………………56 Figure 22: