Metabolic labelling of choline phospholipids probes ABCA3 transport in lamellar bodies |
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| Affiliation: | 1. Department of Pediatric Pneumology, Dr. von Hauner Children''s Hospital, Ludwig-Maximilians University, German Centre for Lung Research (DZL), 80337 Munich, Germany;2. Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children''s Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany;1. Department of Molecular Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea;2. Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea;3. University of Science and Technology (UST), Daejeon 34113, Republic of Korea;4. Department of Physiology, Keimyung University School of Medicine, Daegu 42601, Republic of Korea;5. National Creative Research Initiatives Center for Nuclear Receptor Signals, Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea;6. New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea;7. Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim 105760, Germany;8. Department of Animal Science, College of Agriculture & Life Science, Chonnam National University, Gwangju 61186, Republic of Korea;9. Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu 41944, Republic of Korea;10. Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Republic of Korea;11. Institute for Fundamental Biomedical Research, Departments of Medicine and Biological Chemistry, Johns Hopkins University School of Medicine, St. Petersburg, FL 33701, USA;1. Department of Internal Medicine, Universities of Giessen and Marburg Lung Center, Giessen, Germany;2. Goethe University School of Medicine, Frankfurt am Main, Germany;3. Department of Biochemistry, Universities of Giessen and Marburg Lung Center, Giessen, Germany;1. Laboratory of Immunometabolism and Nutrition, GIGA-Inflammation, Infection & Immunity, University of Liège, Liège, Belgium;2. Division of Diabetes, Nutrition and Metabolic Disorders, Department of Medicine, University Hospital of Liège, Liège, Belgium;3. Laboratory of Lipid Metabolism and Cancer, Department of Oncology, KU Leuven, Leuven, Belgium;4. University of Lille, Inserm, University Hospital of Lille, Pasteur Institute of Lille, U1011 - EGID, F-59000 Lille, France;5. de Duve Institute, Catholic University of Louvain, 1200 Brussels, Belgium;6. Laboratory of Pharmacognosy, CIRM, University of Liège, Liège, Belgium;7. Laboratory of Virology and Immunology, GIGA-Molecular Biology of Diseases, University of Liège, Liège, Belgium |
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| Abstract: | In the metabolism of pulmonary surfactant, the ATP-binding cassette sub-family A member 3 (ABCA3) is a crucial protein in the formation of the storage compartment for surfactant, the lamellar body (LB), and the transport of phospholipids in it. Mutations in ABCA3 not only disturb surfactant metabolism but also cause chronic interstitial lung diseases. Assays for ABCA3 transport function are needed to investigate pathophysiology of the mutations and treatment options for the patients.We metabolically labeled choline (Cho) head phospholipids with the Cho analogue, propargyl-Cho. The universal incorporation of propargyl-Cho was confirmed by mass spectrometry and labeled lipids were visualized in confocal microscopy by click reaction with an azide fluorophore. After pulse-labeling propargyl-Cho labeled lipids accumulated in ABCA3+ vesicles in a time and concentration dependent manner. When treated with the choline kinase inhibitor MN58b during the first 12 h, the lipids intensity inside ABCA3+ vesicles decreased, whereas intensity was unchanged when treated after 12 h. Miltefosine, a substrate of ABCA3, decreased the incorporation of labeled lipids in ABCA3+ vesicles at all time points. The lipids intensity inside the mutated (p.N568D or p.L1580P) ABCA3+ vesicles was decreased compared to wild type, while the intensity outside of vesicles showed no difference.Propargyl-Cho can metabolically pulse-label Cho phospholipids. Visualization and quantification of fluorescence intensity of the labeled lipids inside ABCA3+ vesicles at equilibrium can specifically assess the transport function of ABCA3. |
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