Synthetic processing of surfactant protein C by alevolar epithelial cells. The COOH terminus of proSP-C is required for post-translational targeting and proteolysis.

Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59). Using in vitro transcription/translation, each of the seven constructs produced a 35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events.