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ABCA3 is a surfactant lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ATP-binding cassette, sub-family A (ABC1), member 3 (ABCA3) gene cause respiratory distress syndrome in newborns, and chronic interstitial lung disease in children and adults. ABCA3 belongs to the class of full ABC transporters, which are supposed to be functional in their monomeric forms. Although other family members e.g., ABCA1 and ABCC7 have been shown to function as oligomers, the oligomerization state of ABCA3 is unknown. In the present study, the oligomerization of ABCA3 was investigated in cell lysates and crude membrane preparations from transiently and stably transfected 293 cells using blue native PAGE (BN-PAGE), gel filtration and co-immunoprecipitation. Additionally, homooligomerization was examined in vivo in cells using bioluminescence resonance energy transfer (BRET). Using BN-PAGE and gel filtration, we demonstrate that non-denatured ABCA3 exists in different oligomeric forms, with monomers (45%) and tetramers (30%) being the most abundant forms. Furthermore, we also show the existence of 20% dimers and 5% trimers. BRET analyses verified intermolecular interactions in vivo. Our results also demonstrated that the arrest of ABCA3 in the endoplasmic reticulum (ER), either through drug treatment or induced by mutations in ABCA3, inhibited the propensity of the protein to form dimers. Based on our results, we suggest that transporter oligomerization is crucial for ABCA3 function and that a disruption of oligomerization due to mutations represents a novel pathomechanism in ABCA3-associated lung disease.

Diffuse parenchymal lung diseases (DPLDs) are characterized by chronic inflammation and fibrotic remodeling of the interstitial tissue. A small fraction of DPLD cases can be genetically defined by mutations in certain genes, with ABCA3 being the gene most commonly affected. However, the pathomechanisms underlying ABCA3-induced DPLD are far from clear. To investigate whether ABCA3 plays a role in cellular cholesterol homeostasis, phospholipids, free cholesterol, and cholesteryl esters were quantified in cells stably expressing ABCA3 using mass spectrometry. Cellular free cholesterol and lipid droplets were visualized by filipin or oil red staining, respectively. Expression of SREBP regulated genes was measured using qPCR. Cell viability was assessed using the XTT assay. We found that wild type ABCA3 reduces cellular free cholesterol levels, induces the SREBP pathway, and renders cells more resistant to loading with exogenous cholesterol. Moreover, ABCA3 mutations found in patients with DPLD interfere with this protective effect of ABCA3, resulting in free cholesterol induced cell death. We conclude that ABCA3 plays a previously unrecognized role in the regulation of cellular cholesterol levels. Accumulation of free cholesterol as a result of a loss of ABCA3 export function represents a novel pathomechanism in ABCA3-induced DPLD.

ABCA3 mutations cause fatal surfactant deficiency and interstitial lung disease. ABCA3 protein is a lipid transporter indispensible for surfactant biogenesis and storage in lamellar bodies (LB). The protein folds in endoplasmic reticulum and is glycosylated in Golgi en route to the membrane of mature LB and their precursor multivesicular bodies (MVB). In immunoblots, C-terminally labeled ABCA3 appears as two protein bands of 150 and 190kDa. Using N- and C-terminal protein tags and hindering ABCA3 processing we show that the 150kDa protein represents the mature ABCA3 whose N-terminus is cleaved by a cysteine protease inside MVB/LB.Structured summaryMINT-7996633: Calnexin(uniprotkb:P27824) andABCA3(uniprotkb:Q99758)colocalize(MI:0403) byfluorescence microscopy(MI:0416)MINT-7996380,MINT-7996593,MINT-7996607: LAMP3(uniprotkb:Q9UQV4) andABCA3(uniprotkb:Q99758)colocalize(MI:0403) byfluorescence microscopy(MI:0416)

International audience; ATP-binding cassette transporter A3 (ABCA3) is a lipid transporter active in lung alveolar epithelial type II cells (ATII) and is essential for their function as surfactant-producing cells. ABCA3 mutational defects cause respiratory distress in newborns and interstitial lung disease (ILD) in children. The molecular pathomechanisms are largely unknown; however, viral infections may initiate or aggravate ILDs. Here, we investigated the impact of the clinically relevant ABCA3 mutations, p.Q215K and p.E292V, by stable transfection of A549 lung epithelial cells. ABCA3 mutations strongly impaired expression of the ATII differentiation marker SP-C and the key epithelial cell adhesion proteins E-cadherin and zonula occludens-1. Concurrently, cells expressing ABCA3 mutation acquired mesenchymal features as observed by increased expression of SNAI1, MMP-2 and TGF-beta1, and elevated phosphorylation of Src. Infection with respiratory syncytial virus (RSV), the most common viral respiratory pathogen in small children, potentiated the observed mutational effects on loss of epithelial and acquisition of mesenchymal characteristics. In addition, RSV infection of cells harboring ABCA3 mutations resulted in a morphologic shift to a mesenchymal phenotype. We conclude that ABCA3 mutations, potentiated by RSV infection, induce loss of epithelial cell differentiation in ATII. Loss of key epithelial features may disturb the integrity of the alveolar epithelium, thereby comprising its functionality. We suggest the impairment of epithelial function as a mechanism by which ABCA3 mutations cause ILD.