Probe the transport function of ABCA3 by metabolic labelling of choline phospholipids

Li, Yang (2020): Probe the transport function of ABCA3 by metabolic labelling of choline phospholipids. Dissertation, LMU München: Faculty of Medicine

Abstract

The ATP-binding cassette sub-family A member 3 (ABCA3) plays a critical role in the lipid metabolism of pulmonary surfactant. Locating on the outer membrane of the storage and secretion compartment of surfactant in alveolar type II cells, the lamellar bodies (LBs), ABCA3 is responsible for transporting phospholipids from the cytoplasm into the LBs. Mutations of ABCA3 were identified from patients suffering from neonatal respiratory distress syndrome (RDS) and childhood interstitial lung diseases (chILD). Therefore, exploring the functional assays of ABCA3 is in great demand for pathophysiology investigation and treatment researching. In the present study, wild type and variants of ABCA3 were expressed in A549 epithelial cells to probe mechanisms for transport activity of ABCA3. To quantify the function of ABCA3 alleles, we employed the choline analogue propargyl-Cho. Propargyl-Cho was taken up and the resultant phospholipids can be detected using a combination of click chemistry, fluorescence derivatization and microscopy to visualize and quantify the phospholipid end product. The accumulation of labeled phospholipids was time, concentration dependent. The detection of signal was dependent on choline kinase to incorporate the label into cellular phospholipids. Miltefosine, another substrate of ABCA3 can compete with the transport of propargyl-Cho. Finally, we showed that mutations (p.N568D and p.L1580P) in the ATP-binding cassette of ABCA3 impaired transport function of propargyl-Cho into intracellular vesicles. The present thesis addressed a novel method to assess structure-function relationships of ABCA3.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 600 Technology, Medicine; 600 Technology, Medicine > 610 Medical sciences and medicine
• Faculties: Faculty of Medicine
• Language: English
• Date of oral examination: 17. September 2020
• 1. Referee: Griese, Matthias
• Signature of the printed copy: 0700/UMD 19237