Synthesis and processing of hydrophobic surfactant protein C by isolated rat type II cells.

Surfactant protein C (SP-C) is a 3.7-kDa hydrophobic peptide isolated from organic extracts of pulmonary surfactant which is secreted by alveolar type II cells after synthesis and posttranslational processing of a 21-kDa proSP-C peptide (SP-C 21 ). Previously characterized epitope-specific proSP-C antisera were used to study early proteolytic steps of proSP-C processing by adult rat type II cells. Western blotting and immunocytochemistry using anti-NPROSP-C (epitope = Met 10 -Glu 23 ) each demonstrated marked attenuation of proSP-C protein expression by culture on plastic. Processing was therefore studied by metabolic labeling of freshly isolated type II cells maintained in suspension in serum-free media. With the use of anti-NPROSP-C, immunoprecipitation of cell lysates continuously labeled for 4 h with [ 35 S]methionine demonstrated radiolabeled bands of M r 21, 16, and 10-6,000 while anti-CTERMSP-C (epitope = Ser 149 -Ser 166 ) failed to detect 35 S-bands of M r <16,000. Pulse-chase studies demonstrated synthesis of 35 S-proSP-C 21 with a time-dependent appearance of 16-kDa and 10- to 6-kDa forms which was blocked by addition of brefeldin A. SP-C precursors were not detected in the media. Quantitative analysis of the major bands by direct β-counting indicated a precursor-product relationship between SP-C 21 and SP-C 16 . These results demonstrate the utility of freshly isolated type II cells for characterization of SP-C synthetic pathways and show that early proSP-C processing events include synthesis of a 21-kDa primary translation product followed by extensive intracellular proteolysis of the proSP-C COOH-terminal in subcellular compartments of type II cells which are distal to the trans-Golgi network.