Expression and function of ABCA3 in Type 2 cells

板层小体是II型肺泡上皮细胞的特殊分泌颗粒，细胞通过板层小体包裹表面活性物质并调节其分泌。遗传性表面活性物质蛋白B缺乏导致一种形式的新生儿呼吸窘迫综合征，其特征是板层小体明显减少和形态紊乱。在其他呼吸窘迫的新生儿中也观察到了类似的超微结构表现，其中表面活性蛋白B正常，这意味着其他尚未确定特征的蛋白的异常。板层小体和其他分泌颗粒的形成、维持和利用的过程尚不完全清楚。这个实验室以前的工作已经努力阐明了这些过程。研制了一种识别MR 180 kDa板层体膜独特蛋白的单抗LBM180。对该抗体参与的蛋白质进行了部分质谱测序，结果表明它与ATP结合盒蛋白ABCA3完全相同。这项建议的总体目标是确定ABCA3在II型细胞中的定位、表达和功能。为了实现这一目标，已经或正在积极开发几种试剂，包括全长的人ABCA3 cDNA和正在开发的小鼠和大鼠的cDNA，带有GFP和Myc标签的ABCA3表达载体，ABCA3特异性抗体，以及表达有或没有表位标签的蛋白质或表达反义寡核苷酸的腺病毒构建。抗体将通过免疫荧光和免疫金电子显微镜将ABCA3定位在II型细胞中；这些发现可以通过在适当的II型细胞培养模型中表达标记的ABCA3来证实。这种蛋白质在发育过程中和激素影响下的表达变化将通过Northern和Western blotting来确定。最后，使用反义寡核苷酸和抗体来抑制ABCA3在分化过程中的表达，并在体外干扰其在II型细胞中的功能，以确定ABCA3的功能。由于ABCA3被怀疑是一种磷脂转运体，表面活性物质磷脂组成和II型细胞形态将作为ABCA功能的初步评估。

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.