Expression and function of ABCA3 in type 2 cells

描述（由申请人提供）：片层体（LBs）是2型肺泡上皮细胞（AT2）的分泌颗粒，通过其调节表面活性剂的包装和分泌。我们以前的工作阐明了层状体是如何形成、维持和利用的。制备了一种鉴定层状体膜独特蛋白LBM180的抗体。质谱测序显示LBM180与atp结合盒蛋白ABCA3相同。本研究首次鉴定ABCA3为层状体膜蛋白，并提示其在层状体生物发生中的作用。随后的几项遗传学研究表明，ABCA3基因突变与致命性表面活性物质缺乏和儿童间质性肺疾病有关（Shulenin, 2004; Bullard, 2005）， AT2细胞板层体明显异常（Edwards, 2005）。在动物模型中，我们打算牢固地建立ABCA3突变与肺部疾病之间的因果关系，并开发用于治疗这些疾病的治疗方法的测试方法。在之前的资助期内，我们使用体外方法确定了ABCA3在AT2细胞中的位置和功能。我们发现：1)ABCA3通常被运输到层状体和溶酶体的膜上，并且是将溶酶体转化为层状体所必需的；2)ABCA3对于脂质积累，特别是胆碱磷脂，在层状体和溶酶体中是必要的和充分的；3)破坏细胞中ABCA3表达、运输或功能的突变，原则上可以用小分子疗法来挽救。最近的一项发展使我们能够用一项额外的战略来跟进这些结果。我们从Deltagen生物技术公司购买了5只老鼠，并在宾夕法尼亚大学建立了一个群体。ABCA3-/-小鼠在出生后不久就会死亡，它们的肺不能充气。目的1：我们将检查和分析来自ABCA3+/+、+/-和-/-小鼠肺的AT2细胞形态，并测量ABCA3表达对肺细胞及其分泌产物LB蛋白和脂质含量的影响，以确定ABCA3表达差异如何影响板层体特征。测定细胞培养中ABCA3表达急性变化后的LB含量和PC分泌情况。目的2：使用生化方法，我们将确定ABCA3 atp酶的（脂质）底物。目标3：我们将开发基于细胞筛选的小分子治疗ABCA3突变与运输和折叠缺陷。此外，我们将通过产生ABCA3突变的敲入小鼠和在ABCA3野生型或ABCA3 KO背景下表达条件控制突变型或野生型ABCA3的转基因小鼠来建立新生儿RDS和间质性肺疾病的动物模型。

DESCRIPTION (provided by applicant): Lamellar bodies (LBs) are the secretory granules of the type 2 alveolar epithelial cell (AT2) through which surfactant packaging and secretion are regulated. Our previous work elucidated how lamellar bodies are formed, maintained, and utilized. An antibody identifying a unique protein of the lamellar body membrane, LBM180, was developed. Mass spectrometric sequencing showed LBM180 is identical to the ATP-Binding Cassette protein, ABCA3. This study was the first to identify ABCA3 as a lamellar body membrane, protein and to suggest its role in lamellar body biogenesis. Subsequently several genetics studies showed that mutations of the ABCA3 gene are associated with fatal surfactant deficiency and pediatric interstitial lung disease (Shulenin, 2004; Bullard, 2005) with markedly abnormal lamellar bodies in AT2 cells (Edwards, 2005). In an animal model we intend to firmly establish a causal relationship between ABCA3 mutations and lung disease and develop methods for testing therapeutics for treating these diseases. During the previous funding period we determined the location and function of ABCA3 in AT2 cells using in vitro methods. We showed: 1) ABCA3 is normally trafficked to the membranes of lamellar bodies and lysosomes and is necessary for converting lysosomes to lamellar bodies, 2) ABCA3 is necessary and sufficient for lipid accumulation, especially of choline phospholipids, in lamellar bodies and lysosomes and 3) mutations that disrupt ABCA3 expression, trafficking or function in cells could, in principal, be rescued with small molecule therapeutics. A recent development allows us to follow up these results with an additional strategy. We purchased five mice from Deltagen, a biotechnology company, and established a colony at Penn. The ABCA3-/- mice die soon after birth and their lungs fail to inflate. Aim 1: We will examine and analyze the morphology of AT2 cells from ABCA3+/+, +/- and -/- mouse lungs and measure the effect that ABCA3 expression has on LB protein and lipid content of lung cells and their secreted products to determine how differences in ABCA3 expression affect lamellar body characteristics. The LB content and PC secretion after acute changes of ABCA3 expression in cell cultures will also be measured. Aim 2: Using biochemical approaches we will determine the (lipid) substrates for ABCA3 ATPase. Aim 3: We will develop cell based screens of small molecule therapeutics for ABCA3 mutations with trafficking and folding defects. Further we will develop animal models of neonatal RDS and interstitial lung disease by generating knock-in mice with ABCA3 mutations and transgenic mice expressing conditionally controlled mutant or wild type ABCA3 in a wild type or ABCA3 KO background.