Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome

Abstract

Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures. ATP binding cassette (ABC)2 transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes (1). Among the several subfamilies, the ABCA subclass has received considerable attention, because mutations of the ABCA1 gene cause Tangier disease and mutations of the ABCA4 gene cause Stargardt macular dystrophy in humans (2-5). ABCA1 and ABCA4 are proposed to be transmembrane transporters for intracellular cholesterol/phospholipids and N-retinylidene phosphatidylethanolamine, respectively (3-5). ABCA3, a member of the ABCA subfamily with unknown function (6 -10), is predominantly expressed in the lung and localized to the limiting membrane of lamellar bodies in alveolar epithelial type II cells (ATII) in both humans and rats (7,8).In the lung, development of structures for effective pulmonary gas exchange and production of pulmonary surfactant are necessary for successful adaptation to extrauterine life in the newborn infant. These key processes in lung maturation require differentiation of epithelium into ATII cells, the cellular source for surfactant. Pulmonary surfactant is a complex mixture of lipids, primarily phosphatidylcholine (60 -70% of which is dipalmitoylphosphatidylcholine) and specific proteins that line the alveolar surface of the lung, reducing surface tension at the air-liquid interface and preventing collapse of the lung on expiration (11). Surfactant is assembled and stored in lamellar bodies, the secretory organelles of ATII cells (11-13). Two other members of the ABCA subfamily, ABCA1 and ABCA4, have been implicated in lipid transport leading to the hypothesis that ABCA3 transports lipid into the lamellar bodies of ATII cells (7-9). Recently, it has been reported that mutations in ABCA3 are associated with defective assembly of lamellar bodies and fatal respiratory distress syndrome (RDS) in the newborn infant and interstitial lung disease (6, 10).To study the potential role of ABCA3 in RDS, we examined the subcellular trafficking and substrate specificity of ABCA3 in hATII cells and mammalian cell lines using green fluorescent protein (GFP)-tagged protein and fluorescent lipid analogs. Morphological and functional changes secondary to both loss-and gain-of-function experiments demonstrate that ABCA3 selectively transports phosphatidylcholine, sphingomyelin, and cholesterol to lamellar bodies in hATII cells. Our findings indicate that lipid trafficking by ABCA3 across lamellar body membranes is necessary for lamellar body biogenesis as a key step in assembly of lung surfactant in hATII cells.

Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration

“…23 In vitro, ABCA3 is necessary, but not sufficient for the formation of mature lamellar bodies in alveolar cells, leading to the concept that, in addition to ABCA3, other surfactant proteins such as SP-B are required for the transition of lysosomes to distinct lamellar bodies. 10,24 Besides its function in the lungs, several reports also provided first evidence of a possible role of ABCA3 in cancer cell drug resistance. In a matched pair analysis on a panel of 22 cell lines and their resistant variants on the genomic level, ABCA3 was found to be overexpressed in three resistant variants with an increase in gene copy numbers as the mechanism of ABCA3 abundance.…”

“…The pEGFP-N1-ABCA3 wild-type (wt) and pEGFP-N1-ABCA3 N568D mutant plasmids were described previously. 10 The monoclonal mouse antibody to early endosomal antigen 1 (EEA1) was obtained commercially (Transduction Laboratories), the antibodies to lysosomal-associated membrane protein 1 (LAMP1, code H4A3) and LAMP2 (code H4B4) were from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Human MPR46 was detected using the monoclonal antibody (code 10C6), and human ABCA3 with a polyclonal rabbit antihuman ABCA3 antibody.…”

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

“…Previously, we had functionally characterized a series of disease-related mutations in hABCA3 using similar methodologies ( 5 ). Most prominent of these was L101P, which, when transfected into similar cell lines, produced profound ER retention.…”

“…Previously, we reported that ABCA3 is preferentially traffi cked to lamellar bodies in lung epithelial cells, and to lysosomes and lysosomal-like organelles in A549 and HEK293 cell lines ( 5,21 ). Since the mechanisms governing proper traffi cking of the ABCA3 protein or any of the other ABCA transporters are largely undefi ned, we sought to identify domain(s) mediating their normal biosynthetic routing.…”

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

“…ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells and is proposed to be a surfactant lipid transporter (20,36). Exogenous expression of ABCA3 in cultured cells promotes lipid uptake into intracellular vesicles that generate lamellar body-like vesicles (7,18,21). ABCA3 deficiency in human and mice leads to decreased phosphatidylcholine and phosphatidylglycerol in surfactant, dysgenesis of lamellar bodies, and respiratory distress (1,3,8,11,12,27).…”