Functional characterization of ATP-binding cassette transporter A3 Mutations from Infants with respiratory distress syndrome

Jennifer A. Wambach*, Ping Yang, Daniel J. Wegner, Hillary B. Heins, Lyudmila N. Kaliberova, Sergey A. Kaliberov, David T. Curiel, Frances V. White, Aaron Hamvas, Brian P. Hackett, F. Sessions Cole

*Corresponding author for this work

Research output: Contribution to journalArticle

14 Scopus citations

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 confirmmutation pathogenicity and inform clinical decision making. Our objective was to functionally characterize two ABCA3 mutations (p.R288K and p.R1474W) identified among term and latepreterm 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 wildtype 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.

Original languageEnglish (US)
Pages (from-to)716-721
Number of pages6
JournalAmerican journal of respiratory cell and molecular biology
Volume55
Issue number5
DOIs
StatePublished - Nov 2016

Keywords

  • Childhood interstitial lung disease
  • Neonatal respiratory distress
  • Respiratory distress syndrome
  • Surfactant

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

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    Wambach, J. A., Yang, P., Wegner, D. J., Heins, H. B., Kaliberova, L. N., Kaliberov, S. A., Curiel, D. T., White, F. V., Hamvas, A., Hackett, B. P., & Sessions Cole, F. (2016). Functional characterization of ATP-binding cassette transporter A3 Mutations from Infants with respiratory distress syndrome. American journal of respiratory cell and molecular biology, 55(5), 716-721. https://doi.org/10.1165/rcmb.2016-0008OC