Trafficking of newly synthesized surfactant protein B to the lamellar body in alveolar type II cells
Trafficking of newly synthesized surfactant protein B to the lamellar body in alveolar type II cells
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DOI：
10.1007/s00441-020-03232-7
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发表时间：
2020-06
影响因子：
3.6
通讯作者：
K. Osanai;S. Mizuno;H. Toga;Keiji Takahashi
中科院分区：
文献类型：
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作者：
K. Osanai;S. Mizuno;H. Toga;Keiji Takahashi
肺表面活性物质在板层体（LB）中不仅通过分泌（顺行）途径积累，而且通过内吞（逆行）途径积累。我们之前的研究表明，表面活性剂的主要成分磷脂酰胆碱和表面活性剂蛋白A在肺泡II型细胞中有独立的转运途径。因此，表面活性剂蛋白B (SP-B)，一种主要的疏水表面活性剂载脂蛋白，应该用一种直接的方法重新评估。通过对细胞进行放射性标记和随后的细胞分离，研究新合成的SP-B在兔肺泡II型细胞中的序贯转运。采用梯度超离心法制备LB馏分。用抗sp - b抗体对培养基、总细胞和LB部分进行免疫沉淀。1 h后在细胞中检测到新合成的[35S]-pro-SP-B (~ 42 kDa)， 3 h后在细胞中检测到成熟的[35S]-SP-B， 6 h后在LB中检测到成熟的[35S]-SP-B， 24 h后在细胞中以成熟的[35S]-SP-B为主，优势部分存在于LB中，而培养基中只有少量成熟的[35S]-SP-B。~ 42 kDa [35S]-pro-SP-B的分子加工和向LB的运输被分解高尔基体的brefeldin A所抑制。这些结果表明，新合成的SP-B通过高尔基体分选到LB，并储存到胞吐。该途径与报道的磷脂酰胆碱和表面活性剂蛋白A的途径不同。
Lung surfactant accumulates in the lamellar body (LB) via not only the secretory (anterograde) pathway but also the endocytic (retrograde) pathway. Our previous studies suggested that the major surfactant components, phosphatidylcholine and surfactant protein A take independent trafficking routes in alveolar type II cells. Thus, trafficking of surfactant protein B (SP-B), a major hydrophobic surfactant apoprotein, should be re-evaluated by a straightforward method. Radiolabeling of cells and subsequent cell fractionation were employed to pursue the sequential trafficking of newly synthesized SP-B in rabbit alveolar type II cells. The LB fraction was prepared by gradient ultracentrifugation. Immunoprecipitation from the culture medium, total cells, and LB fraction was carried out with anti-SP-B antibody. Newly synthesized [35S]-pro-SP-B (~ 42 kDa) was detected in the cells after 1 h. An ~ 8-kDa mature form of [35S]-SP-B was detected in the cells after 3 h and in the LB after 6 h. Mature [35S]-SP-B was predominant in the cells after 24 h, and the dominant portion was present in the LB. In contrast, only a small amount of mature [35S]-SP-B was present in the culture medium. Molecular processing of ~ 42 kDa [35S]-pro-SP-B and transport to the LB was inhibited by brefeldin A, which disassembles the Golgi apparatus. These results suggest that newly synthesized SP-B is sorted to the LB via the Golgi and stored until exocytosis. This pathway is distinct from the pathways reported for phosphatidylcholine and surfactant protein A.