Expression and function of ABCA3 in Type 2 cells

ABCA3在2型细胞中的表达和功能

• 批准号: 6569883
• 负责人: HENRY SHUMAN
• 金额: $ 24.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-10 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6569883
• 关键词: adenosine triphosphate; animal tissue; antisense nucleic acid; cell differentiation; granule; human fetus tissue; immunoelectron microscopy; immunofluorescence technique; immunologic assay /test; intracellular membranes; membrane proteins; membrane transport proteins; phospholipids; protein localization; pulmonary surfactants; respiratory epithelium; tissue /cell culture; transfection; transfection /expression vector

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.

片层小体是II型肺泡上皮细胞的特化分泌颗粒，细胞通过片层小体包裹表面活性剂并调节其分泌。遗传性表面活性剂蛋白B缺乏引起新生儿呼吸窘迫综合征的一种形式，其特征是板层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类似的超微结构外观，其中表面活性剂蛋白B正常，这意味着其他尚未表征的蛋白质异常。层状体和其他分泌颗粒形成、维持和利用的过程尚不完全清楚。这个实验室以前的工作一直在努力阐明这些过程。制备了一种单克隆抗体，用于鉴定mr180kda片层体膜的独特蛋白LBM180。该抗体参与的蛋白质的部分质谱测序表明，它与atp结合的Cassette蛋白ABCA3相同。本提案的总体目的是确定ABCA3在II型细胞中的定位、表达和功能。为了实现这一目标，已经开发或正在积极开发几种试剂，包括全长人ABCA3 cDNA和正在开发的小鼠和大鼠cDNA，带GFP和Myc标签的ABCA3表达载体，ABCA3特异性抗体，以及用于表达带或不带表位标签或表达反义寡核苷酸的腺病毒构建物。抗体可以通过免疫荧光和免疫金电镜在II型细胞中定位ABCA3；这些发现可以通过在适当的II型细胞培养模型中标记ABCA3的表达来证实。该蛋白在发育过程中以及在激素影响下的表达变化将通过Northern和Western blots测定。最后，利用反义寡核苷酸和抗体测定ABCA3的功能，在体外II型细胞中抑制其在分化过程中的表达并干扰其功能。由于ABCA3被怀疑是一种磷脂转运体，表面活性剂磷脂组成和II型细胞形态将首先作为ABCA功能的测定来评估。