Adenosine triphosphate (ATP)-binding cassette A3 transporter (ABCA3) is a member of the evolutionarily highly conserved family of ABC transporters. The ABCA3 gene (more than 80 kb length) maps to human chromosome 16 (16p13.3) and consists of 33 exons (30 of which are coding). ABCA3 encodes a 1704 amino acid (~150 kDa) protein (Dean 2005). ABCA3 is a lung-specific phospholipid transporter with a critical role in the intracellular surfactant synthesis and storage in lamellar bodies (LBs), which is organized into two tandem functional units (N-half and C-half), consisting in a TMD (α-helix motif) and a NBD for each half (Bruder et al. 2007; Park et al. 2010). The expression of ABCA3 is developmentally regulated, peaking prior to birth under the influence of steroids and transcription factors. Bi-allelic mutations of the ABCA3 gene represent the most frequent cause of congenital surfactant deficiency, indicating its critical role in lung function (Shulenin et al. 2004).
Functional Features of ABC Transporters
In eukaryotes, ABC transporters are located in the plasma membrane, in the membranes of intracellular compartments such as the Golgi, endosome, multivesicular bodies, endoplasmic reticulum, peroxisome, and in mitochondria. To date, 48 members of the human family of ABC transporters have been identified. Based on their structural relatedness, these members are subdivided into seven families, designated ABC A–G (Dean and Annilo 2005).
Structural Features of ABCA3
Adenosine triphosphate (ATP)-binding cassette A3 transporter (ABCA3) is a transmembrane lipid carrier that belongs to the ABC transporter family. Twelve human ABC transporter genes belonging to the A-subfamily (ABCA1–10, ABCA12, and ABCA13) and two pseudogenes (ABCA11P and ABCA17P) are known so far (Dean 2005; Paolini et al. 2015; Piehler et al. 2006). ABCA3 is a 150 kDa, 1704 amino acid protein encoded by an 80 kb gene composed of 33 exons, only 30 of which are transcribed, located at the 16p13.3 genomic locus. ABCA3 is organized into two tandem functional units (N-half and C-half), consisting in a TMD (α-helix motif) and a NBD for each half (Bruder et al. 2007; Park et al. 2010). Both N- and C-halves of ABCA3 have been predicted to contain a TMD formed by six hydrophobic membrane-spanning α-helices connected by a long loop (156–220 aa) connecting the first and the second α-helix and by short hydrophilic loops (8–53 aa length) linking the remaining helices of the TMS. The extracellular domain (ECD) loops and the TMDs constitute the main substrate binding site that allows the trafficking of lipid molecules. As in other ABCA proteins, ABCA3 presents a short (~22 aa) N-terminal protein segment of positively charged residues, which is followed by the first α-helix of the transmembrane motif: a domain rich in hydrophobic amino acids such as valine and leucine. The extracellular domains (ECDs) between the first α-helix (N- and C-half) and the rest of the TMD domain protrude in the extracellular compartment. However, in ABCA3 protein, the function of these two extracellular loops is still unknown (Vedhachalam et al. 2007).
Most of the members of ABC transporters have a similar domain organization except for the number of transmembrane domains that may vary (i.e., ABCB2, ABCC1, etc.) or the different length of the loops that connect them. The ABCA3 nucleotide binding domains (NBD1 and NBD2) consist of 153 and 154 amino acids, respectively, and are generally conserved in many other species (Higgins 1992).
Expression, Regulation, and Function of ABCA3
The ABCA3 protein can be readily detected by 26–27 weeks of gestation in normal fetuses, but as early as 23–24 weeks when lung inflammation is present. Its expression increases in the presence of lung infection and bronchopulmonary dysplasia (Stahlman et al. 2007; Yoshida et al. 2004). Several factors and hormones regulate ABCA3 expression. Among the most important, we recall the glucocorticoids, such as dexamethasone (Yoshida et al. 2004), thyroid transcription factor 1 (TTF-1) which play a switch role in epithelial maturation and surfactant synthesis inducing the expression of ABCA3, SP-A, SP-B, and SP-C (Stahlman et al. 2007; Yoshida et al. 2004; Kolla et al. 2007) and STAT3 (signal transducer and activator of transcription 3) that induces ABCA3 expression and possibly mediates its response to inflammation (Matsuzaki et al. 2008). Other enhancing ABCA3 transcription factors include FOXA2 (forkhead box A2), GATA6 (GATA-binding protein 6), and NFATc3 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3) (Besnard et al. 2007). Finally, ABCA3 transcription is also inducible by SREBP1a and SREBP1c (sterol regulatory element-binding transcription factor 1a/c) proteins that contain several sterol-responsive elements and are involved in lamellar bodies formation (Besnard et al. 2007).
ABCA3 expression was detected at highest levels in type II alveolar epithelial cells, but it was also found in other organs including liver, stomach, kidney, adrenal, pancreas, trachea, and brain. Specifically, ABCA3 has been localized predominantly to the limiting membrane of lamellar bodies, lipid-rich organelles associated with the production, storage, and secretion of pulmonary surfactant (Yamano et al. 2001). Vesicular structures observed in HEK293 cells exogenously expressing ABCA3 suggest the involvement of ABCA3 protein in the generation of lamellar body-like structures (Nagata et al. 2004). Further evidence supporting the hypothesis that ABCA3 might be involved in assembly of lung surfactant were provided by in vitro studies, suggesting that ABCA3 transport of phospholipids and cholesterol is essential for lamellar body formation, a key step in assembly of lung surfactant, in primary cultures of human AT2 cells and the human carcinoma A549 cell line (Cheong et al. 2006).
ABCA3 Deficiency and Mechanisms of Disease
ABCA3 deficiency is appearing to be one of the most common cause of inborn errors of surfactant metabolism. More than 180 distinct mutations including multiple-missense, splice-site, and frameshift mutations in the gene encoding ABCA3 have been found in children with severe neonatal respiratory disease and adolescents with some forms of interstitial lung disease (Shulenin et al. 2004). In humans, mutations in the ABCA3 gene are associated with decreased surfactant phosphatidylcholine content and increased surface tension of the lung lavage fluid, also suggesting that ABCA3 plays an important role in pulmonary surfactant phospholipid homeostasis (Garmany et al. 2006). Since the phenotype of mice with targeted disruption of the ABCA3 gene was similar to that of humans with functional or trafficking mutations in the ABCA3 gene, the use of animal models has contributed to increase the knowledge regarding the physiologic transport function of ABCA3. Engineered Abca3−/− mice that completely lack ABCA3 expression die of respiratory failure as a result of an inability to secrete pulmonary surfactant into the alveolar space (Fitzgerald et al. 2007). Furthermore, it has been recently demonstrated that adult mice with ABCA3 haplo-insufficiency are more susceptible to the deleterious effects of lung injury by hyperoxia or mechanical ventilation (Herber-Jonat et al. 2013). However, the wide spectrum of mutations in the ABCA3 gene well correlate with different phenotype forms of severe respiratory distress, such as persistent or recurrent tachypnea in newborns (Kinane et al. 2011; Prestridge et al. 2006), and mutations associated with milder clinical forms, such as ILD (interstitial lung disease), desquamative interstitial pneumonitis presenting in childhood (Whitsett et al. 2010), and idiopathic pulmonary fibrosis or emphysema in adults (Epaud et al. 2014). ABCA3 deficiency was characterized to variable histopathology showing combinations of alveolar epithelial type 2 cells (AEC2) hyperplasia, intraalveolar eosinophilic material, alveolar macrophages, desquamated epithelial cells, interalveolar septa thickening, fibrosis, and inflammation (Brasch et al. 2006; Citti et al. 2013). Ultrastructural anomalies, observed following TEM analysis, put in evidence a profound disruption of surfactant composition and function due to decreased amount of phosphatidylcholine and other phospholipids that induce high alveolar surface tension values compared with controls and to surfactant protein B (SP-B) deficiency (Garmany et al. 2006; Edwards et al. 2005). Moreover, altered SP-B and surfactant protein C (SP-C) processing and routing occur also in ABCA3 deficiency and probably contribute to surfactant homoeostasis disruption and respiratory symptoms.
This pathological scenario has become more complex, and recently several studies have demonstrated that the surfactant dysfunction and respiratory disease not only correlate with mutations located in the exons or the immediate intron-exon boundaries, but also with noncoding regions, resulting in aberrant ABCA3 mRNA splicing and expression (Agrawal et al. 2012).
The uniqueness and variability of ABCA3 variants overlapped to other surfactant-related genetic diseases strongly support the use of high-throughput approaches, such as next generation sequencing or whole-exome sequencing to get a more comprehensive picture of ABCA3 deficiencies (Wambach et al. 2014). Mutation type and position, in silico validation tools, human variation database, species conservation databases, and inheritance patterns should be complemented by strict phenotypical characterization. Although lung transplantation remains the only option for end-stage lung disease, given its poor long-term prognosis, some less severe forms of ILD may benefit from pharmacological treatment. Inflammatory modulating agents, such as pulse methylprednisolone, hydroxychloroquine, and azithromycin, are used occasionally either individually or in combination (Flamein et al. 2012), although their use has not been validated by proper clinical trials. A deeper understanding of the pathogenic mechanisms could lead to the development of mutation-specific drugs, such as proteasome inhibitors in mutations affecting glycosylation site (Beers et al. 2013) or chemical chaperones in those affecting intracellular trafficking (Cheong et al. 2006).
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