Lamellar bodies are specialized secretory granules of the type II alveolar epithelial cell through which the cell packages surfactant and regulates its secretion. Hereditary surfactant protein B deficiency causes a form of respiratory distress syndrome of the newborn characterized by marked diminution of lamellar bodies and derangement in their morphology. A similar ultrastructural appearance has been observed in other neonates with respiratory distress in which surfactant protein B is normal, implicating abnormalities in other, as of yet uncharacterized proteins. The processes by which lamellar bodies and other secretory granules are formed, maintained, and utilized are incompletely understood. Previous work in this laboratory has endeavored to elucidate these processes. A monoclonal antibody identifying a unique protein of the lamellar body membrane of Mr 180kDa, LBM180, was developed. Partial mass spectrometric sequencing of the protein participated by this antibody indicates that it is identical to the ATP-binding Cassette protein, ABCA3. The General Aim of this proposal is to determine the localization, expression, and function of ABCA3 in type II cells. Several reagents have already been developed or are actively being developed to accomplish this goal, including the full-length human ABCA3 cDNA and ongoing development of the mouse and rat cDNAs, ABCA3 expression vectors with GFP and Myc tags, ABCA3- specific antibodies, and adenoviral constructs to express the protein with or without epitope tags or to express antisense oligonucleotides. Antibodies will enable localization of ABCA3 within type II cells by immunofluorescence and immuno-gold electron microscopy; these findings can be confirmed by expression of tagged ABCA3 in appropriate type II cell culture models. Changes in expression of this protein during development and under the influence of hormones will be determined using Northern and Western blots. Finally, the function of ABCA3 will be determined using antisense oligonucleotides and antibodies to inhibit its expression during differentiation and perturb its function in type II cells in vitro. Since ABCA3 is suspected to be a phospholipid transporter, surfactant phospholipid composition and type II cell morphology will be assessed initially as assays of ABCA function.
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