Classification of different ABCA3 mutations causing interstitial lung disease

Abstract

Introduction: ABCA3 is a lipid transporter found in alveolar type II cells, where it localizes to the outer membrane of lamellar bodies which store surfactant. Mutations in the ABCA3 gene are the most common known genetic cause of respiratory distress syndrome in newborns and of late onset interstitial lung disease in children. Objectives and methods: WT ABCA3 cDNA fused to a HA-tag was cloned into the pT2HB SB expression vector. Clinical relevant mutations R208W, Q215K, R288K, E292V, M760R, G964D and K1388N were inserted by site-directed mutagenesis into the WT ABCA3 gene. Vectors were stably transfected into A549 cells with the “Sleeping Beauty” system based on transposition. The level of ABCA3 expression and processing was examined by quantitative PCR and immunoblot analysis. Subcellular protein localization was analyzed by immunofluorescence. Changes in total lipid composition within the different cell lines were investigated by mass spectrometry. Results: Immunoblot analyses of mutated ABCA3 revealed changes in protein processing compared to the wildtype. Furthermore we found that ABCA3 is localized mainly at the limiting membrane of CD63-positive vesicles, whereas Q215K and M760R showed a different localization pattern. Mass spectrometry analyzed indicated alteration of lipid composition. Conclusion: We have prepared and characterized cellular models with ABCA3 mutations in order to investigate specific mechanisms for restoring its wildtype function. Supported by “Deutsches Zentrum fur Lungenforschung“ (DZL) and „Deutsche Forschungsgemeinschaft“ (DFG).