Divergent pathways of surfactant protein C maturation for disease-associated isoforms

Surfactant protein C (SP-C), a hydrophobic protein exclusively synthesized and secreted by alveolar type II (AT2) cells, is important for reducing alveolar surface tension in the distal lung. Chronic interstitial pulmonary diseases have been associated with SFTPC mutations. However, a detailed understanding of SP-C maturation in the secretory pathway and disruptions caused by mutations has remained incomplete. The goal of this study was to comprehensively ascertain differences in trafficking and posttranslational processing between WT and disease-associated SP-C mutants using doxycycline-inducible mouse lung epithelial cell lines expressing either WT SP-C or the common clinical variant SP-C^I73T, validated using primary AT2 cells isolated from a murine SP-C^I73T pulmonary fibrosis model and induced pluripotent stem cell-derived human AT2 cells expressing the same mutant. In all three models SP-C^WT was highly concentrated in acidic lysosomal-related organelles while SP-C^I73T accumulated on the plasma membrane, which was corroborated by inhibition of clathrin-mediated endocytosis, surface biotinylation, immunogold electron microscopy, immunofluorescent staining, and proteinase K protection assays supporting divergence of SP-C^I73T trafficking from SP-C^WT. The exclusion of SP-C^I73T from normal routing occurred early in the biosynthetic pathway as brefeldin A blocked processing of both SP-C proproteins, while a 20 ˚C temperature shift caused selective accumulation of a processed proSP-C^WT intermediate, suggesting initial C-terminal cleavage of proSP-C^WT occurs in late-Golgi/trans-Golgi network. This cleavage event was sensitive to DC1, an inhibitor of furin-related subtilisin-like proprotein convertase (PPC) family members. Site-directed mutagenesis of canonical residues K160/R167 within a predicted PPC recognition site in the proSP-C COOH domain blocked its processing. Expression constructs encoding inhibitory pre-proprotein peptide fragments of furin and PC7 each inhibited cleavage of proSP-C^WT in mouse lung epithelial-12 cells. Collectively, our data demonstrate that trafficking pathways for maturation of WT and mutant I73T SP-C diverge prior to the trans-Golgi network, where initial cleavage of the COOH-terminal SP-C propeptide occurs via a furin-like proprotein convertase.