Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific...

Surfactant protein C (SP-C), a 3.7-kDa hydrophobic lung-specific protein, is synthesized and secreted by pulmonary type II cells through proteolytic processing of a 21-kDa propeptide (SP-C21) by currently undefined pathways. Previously, we reported the production of a polyclonal antibody against rat SP-C21 (anti-CPROSP-C) using a synthetic peptide as the immunizing antigen (Beers, M. F., Wali, A., Eckenhoff, M. E. F., Feinstein, S., Fisher, J. H., and Fisher, A. B. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 368-378). In this study, two additional epitope-specific proSP-C antibodies produced using synthetic peptide sequences were utilized to examine synthetic processing of SP-C. Anti-NPROSP-C (Met10-Gln23) and anti-CTERMSP-C (Ser149-Ser166) recognized native proSP-C21 produced from in vitro translation of SP-C cDNA. Immunocytochemistry using anti-NPROSP-C confirmed the localization of proSP-C peptides exclusively in type II cells. Western analysis of subcellular fractions identified a single 21-kDa band in microsomes and a 16-kDa form in lamellar bodies each recognized by all three antisera, while anti-NPROSP-C also uniquely identified a prominent 5-6-kDa form in lamellar bodies. In a perfused rat lung model labeled with [35S]cysteine/methionine, immunoprecipitation of lung homogenate and lamellar body fractions identified early appearances of 35S-labeled 21-, 18-, and 16-kDa SP-C forms in homogenate and a 16-kDa intermediate form in lamellar bodies. Anti-NPROSP-C also exclusively detected time-dependent appearances of 5-10-kDa proSP-C forms in lamellar bodies and homogenates. Processing of proSP-C21 was completely blocked by inclusion of brefeldin A (15 micrograms/ml) in the perfusate. These results demonstrate that synthetic peptides can be used to produce epitope-specific antisera which recognize more hydrophilic domains of proSP-C and show that proSP-C processing occurs intracellularly in subcellular compartments of type II cells which are distal to the trans-Golgi network.