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

小GTP结合蛋白通过参与细胞信号传导途径介导许多必需的细胞内功能。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文库中克隆出来并记录在Genbank中，但其功能在很大程度上仍然未知。重组蛋白在昆虫细胞中表达，表达的重组杆状病毒携带多聚组氨酸标记的rab p24 cDNA，并通过镍亲和层析纯化。用从rab p24的C-末端氨基酸序列命名的合成肽产生抗兔的多克隆抗体。Western blot显示，使用抗体的特异性免疫反应带对应于预测的分子量的rab p24在总肺匀浆和分离的肺泡II型细胞，但没有发现在肺泡巨噬细胞匀浆。冰冻大鼠肺组织的免疫染色显示气道上皮细胞和肺泡角细胞（可能是肺泡II型细胞）的阳性信号。肺泡Ⅱ型细胞涂片免疫组化染色阳性，而肺泡巨噬细胞涂片免疫组化染色阴性。纯化的肺泡Ⅱ型细胞总RNA RT-PCR阳性，肺泡巨噬细胞RT-PCR阴性。RNA核糖核酸探针原位杂交显示肺泡角细胞和气道上皮细胞阳性，尤其是终末气道细胞（其中Clara细胞占80%以上）阳性。特异性阳性免疫反应信号见于分离的肺泡II型细胞的重囊泡组分的裂解物中，但未见于板层体组分中。这些结果表明rabp24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，即肺表面活性物质代谢，该蛋白可能在表面活性物质转运中起作用。

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.