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Trafficking of newly synthesized surfactant protein B to the lamellar body in alveolar type II cells

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Abstract

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.

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Abbreviations

SP-B:

Surfactant protein B

LB:

Lamellar body

BFA:

Brefeldin A

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Acknowledgments

The mouse anti-pig SP-B monoclonal antibody (8B5E) and the rabbit anti-rat SP-A antibody (1D6) were generous gifts from Dr. Yasuhiro Suzuki (Department of Ultrastructural Research, Institute for Frontier Medical Sciences, Kyoto University) and Dr. Dennis R. Voelker (National Jewish Health, Denver, CO), respectively.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (C) (18K08191) from The Ministry of Education, Science, Sports, and Culture, Japan.

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Authors and Affiliations

  1. Department of Life Science, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku-Uchinada, Kahoku-gun, Ishikawa, 920-0293, Japan

    Kazuhiro Osanai

  2. Department of Respiratory Medicine, Kanazawa Medical University, 1-1 Daigaku-Uchinada, Kahoku-gun, Ishikawa, 920-0293, Japan

    Kazuhiro Osanai, Shiro Mizuno, Hirohisa Toga & Keiji Takahashi

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  1. Kazuhiro Osanai
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  2. Shiro Mizuno
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Correspondence to Kazuhiro Osanai.

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Osanai, K., Mizuno, S., Toga, H. et al. Trafficking of newly synthesized surfactant protein B to the lamellar body in alveolar type II cells. Cell Tissue Res 381, 427–438 (2020). https://doi.org/10.1007/s00441-020-03232-7

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