Categorization and classification of different ABCA3 variants causing interstitial lung disease

Abstract

ATP-binding cassette subfamily A member 3 (ABCA3) is a lipid transporter found in type II pneumocytes, where it localizes to the outer membrane of lamellar bodies (LBs). LBs derive from lysosomal origin and are essential for storing phospholipids and surfactant. Mutations in the ABCA3 gene are the most common known genetic cause of respiratory distress syndrome (RDS) in newborns and of late onset interstitial lung disease (ILD) in children. But the effects of most variations on ABCA3 protein function are still poorly understood. Therefore we looked in detail at different sequence variations of ABCA3 and their impact on wild type function. The R288K variation of ABCA3 was found to have an increased frequency in a population of patients suffering from ILD. These patients did not exhibit characteristic features of complete ABCA3 deficiency. Nevertheless, we found strong evidence that R288K variation affects transport function of ABCA3 protein, supported by decelerated detoxification of doxorubicin, reduced dipalmitoyl-phosphatidylcholine (PC 32:0) content, and decreased LB volume. By contrast, the K1388N variation recently found in a patient suffering from ILD with lethal outcome led to complete ABCA3 deficiency. We showed that this sequence variation of ABCA3 correctly localized to LBs but had a processing defect and a reduced lipid transporter activity, proven by reduced PC 32:0 content and malformed LBs. Due to existent intracellular processing defects of K1388N sequence variation and other mutations, we particularly looked at potential protease(s) cleaving ABCA3. An identification of these specific proteins may represent a potential therapeutic target. We found that ABCA3 is proteolytically cleaved by cathepsin L and to a lower level by cathepsin B. Furthermore, we identified the exact cleavage site of cathepsin L located after Lys174 in the ABCA3 protein. In summary, the molecular tools used in these studies, together with a close correlation of our in-vitro and ex-vivo data will allow a better knowledge of ABCA3 wild type function. Besides this, groups of mutations with similar molecular defects can be defined and targeted by specific small molecule correctors for restoring impaired ABCA3 transporter function in the future.