Functional Characterization of ATP-Binding Cassette Transporter A3 Mutations from Infants with Respiratory Distress Syndrome

Abstract

Mutations in the ATP-binding cassette transporter A3 gene (ABCA3) result in severe neonatal respiratory distress syndrome and childhood interstitial lung disease. As most ABCA3 mutations are rare or private, determination of mutation pathogenicity is often based on results from in silico prediction tools, identification in unrelated diseased individuals, statistical association studies, or expert opinion. Functional biologic studies of ABCA3 mutations are needed to confirm mutation pathogenicity and inform clinical decision making. Our objective was to functionally characterize two ABCA3 mutations (p.R288K and p.R1474W) identified among term and late-preterm infants with respiratory distress syndrome with unclear pathogenicity in a genetically versatile model system. We performed transient transfection of HEK293T cells with wild-type or mutant ABCA3 alleles to assess protein processing with immunoblotting. We used transduction of A549 cells with adenoviral vectors, which concurrently silenced endogenous ABCA3 and expressed either wild-type or mutant ABCA3 alleles (p.R288K and p.R1474W) to assess immunofluorescent localization, ATPase activity, and organelle ultrastructure. Both ABCA3 mutations (p.R288K and p.R1474W) encoded proteins with reduced ATPase activity but with normal intracellular localization and protein processing. Ultrastructural phenotypes of lamellar body-like vesicles in A549 cells transduced with mutant alleles were similar to wild type. Mutant proteins encoded by ABCA3 mutations p.R288K and p.R1474W had reduced ATPase activity, a biologically plausible explanation for disruption of surfactant metabolism by impaired phospholipid transport into the lamellar body. These results also demonstrate the usefulness of a genetically versatile, human model system for functional characterization of ABCA3 mutations with unclear pathogenicity.

Keywords: childhood interstitial lung disease; neonatal respiratory distress; respiratory distress syndrome; surfactant.

Figures

Figure 1.

(A and B) Construction of adenoviral expression cassettes. cDNA, complementary DNA; GFP, green fluorescent protein; shRNA, short hairpin RNA; SV40, Simian virus 40.

Figure 2.

(A and B) Intracellular immunofluorescent localization of wild-type (WT) ABCA3_GFP and mutant proteins. A549 cells transfected with WT ABCA3 or mutations, p.L101P, p.E292V, p.R288K, and p.R1474W, were analyzed using confocal microscopy. Anti-CD63 and anti-calnexin were used to stain lysosomally derived lamellar body–like vesicles and endoplasmic reticulum, respectively. Scale bars, 5 μm (n = 3 replicates per condition). ABCA3, ATP-binding cassette transporter A3; EGFP, enhanced green fluorescent protein.

Figure 3.

Immunoblotting analysis in cells transiently transfected with WT ABCA3 or p.L101P, p.E292V, p.R288K, and p.R1474W mutations. ABCA3 expression was detected using anti-GFP antibody and normalized with β-actin (n = 3 replicates per conditions).

Figure 4.

ATPase activity of WT ABCA3 and mutant proteins. ATPase activity was measured as free v-phosphate (Pi) released, compared with WT ABCA3 activity, and normalized to Western blot with anti-GFP antibody. Error bars represent the mean ± SD (n = 3 replicates per condition). All three mutant proteins had significantly reduced ATPase activity compared with WT ABCA3. *P < 0.0001; †P = 0.0012.

Figure 5.

Electron microscopy imaging of A549 cells transduced with WT ABCA3, p.L101P, p.E292V, p.R288K, and p.R1474W. A549 cells transduced with p.E292V, p.R288K, and p.R1474W appeared similar to WT with multiple, well-organized lamellar body–like vesicles. Cells transduced with p.L101P had frequent abnormal-appearing lamellar body–like vesicles with dense inclusions and fewer well-organized lamellar body–like vesicles compared with cells transduced with WT. White arrows indicate well-organized lamellar body–like vesicles; black arrows indicate abnormal-appearing lamellar body–like vesicles; white arrowhead indicates lamellar body–like structure in untransduced cells; black arrowheads indicate mitochondria. Scale bar, 0.5 μm (n = 10 cells examined per condition).