Metabolic labelling of choline phospholipids probes ABCA3 transport in lamellar bodies

Li, Yang; Kinting, Susanna; Hoeppner, Stefanie; Forstner, Maria Elisabeth; Uhl, Olaf; Koletzko, Berthold und Griese, Matthias (2019): Metabolic labelling of choline phospholipids probes ABCA3 transport in lamellar bodies. In: Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids, Bd. 1864, Nr. 12, UNSP 158516

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 1388-1981
• Sprache: Englisch