Investigation of cell biological function of a small G protein expressed in alveolar type II cells

小的GTP结合蛋白通过参与细胞信号通路来调节一些基本的细胞内功能。我们研究了一种新的Rab蛋白在肺组织中的表达和定位。该蛋白的cDNA已被其他研究人员从大鼠肺cDNA文库中克隆出来，并在Genbank中被记录，但其功能在很大程度上仍不清楚。用携带多组氨酸标记的Rab p24基因的重组杆状病毒在昆虫细胞中表达重组蛋白，并用镍亲和层析进行纯化。用Rab p24的C端氨基酸序列合成的多肽，制备了兔多克隆抗体。免疫印迹显示，在肺匀浆和分离的肺泡II型细胞中，Rab p24的相对分子质量与预测的免疫活性条带一致，而在肺泡巨噬细胞匀浆中未发现。冰冻大鼠肺组织免疫染色显示，在呼吸道上皮细胞和肺泡角细胞(可能是肺泡II型细胞)有阳性信号。分离的肺泡II型细胞涂片免疫染色阳性，而肺泡巨噬细胞涂片免疫染色阴性。从纯化的肺泡II型细胞中提取的总RNA RT-PCR为阳性，而肺泡巨噬细胞的总RNA为阴性。用RNA核探针原位杂交显示肺泡角细胞和呼吸道上皮细胞呈阳性信号，尤其是在终末气道细胞(在大鼠中80%以上是Clara细胞)。分离的肺泡II型细胞的重泡部分的裂解液中可见特异性阳性免疫反应信号，而板层小体部分的裂解液中未见特异性阳性免疫反应信号。这些结果表明，Rab p24定位于Clara细胞和肺泡II型细胞。由于这些细胞具有不同的功能，即肺表面活性物质的代谢，该蛋白可能在表面活性物质的运输中发挥作用。

Small GTP-binding proteins mediate a number of essential intracellular functions via participating' in cell signaling pathways. We investigated expression and localization of a novel rab protein in the lung tissue. cDNA of the protein had been cloned out from a rat lung cDNA library and recorded in Genbank by other researchers, but its function remains largely unknown. Recombinant protein was expressed in insect cells transfected with recombinant baculovirus carrying polyhistidine-tagged rab p24 cDNA, and was purified with a nickel affinity chromatography. Polyclonal antibody was raised against rabbits with a synthetic peptide designated from C-terminal amino acid sequence of the rab p24. Western blot using the antibody showed specific immunoreactive band corresponding to the predicted molecular weight of the rab p24 in total lung homogenate and isolated alveolar type II cells, but was not found in alveolar macrophage homogenate. Immunostaining of a frozen rat lung tissue revealed positive signal in airway epithelial cells and alveolar corner cells (possibly alveolar type II cells). Cell smear of isolated alveolar type II cells was also positive in immunostaining, whereas that of alveolar macrophages was negative. RT-PCR of total RNA extracted from purified alveolar type II cells was positive, whereas that of alveolar macrophages was negative, in situ hybridization using RNA riboprobe showed positive signals in alveolar corner cells and airway epithelial cells, especially in terminal airway cells (of which more than 80% are Clara cells in rats). Specific positive immunoreactive signal was seen in lysate of heavy vesicle fraction from isolated alveolar type II cells but was not seen in that of lamellar body fraction. These results showed that the rab p24 localizes in Clara cells and alveolar type II cells. Since these cells have distinct function, i.e. pulmonary surfactant metabolism, the protein may have a role in surfactant transport.