Interstitial lung disease-associated isoforms induce divergent pathways of maturation for Surfactant Protein C biosynthesis

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. In vivo, the 21-kDa proSP-C undergoes sequential C-term and N-term cleavages through 16-, 7-, and 6-kDa intermediates to generate mature 4.2-kDa SP-C. While Cathepsin H and AT2-specific enzymes such as Napsin/Pepsinogen C are implicated in late N-terminal processing within lamellar bodies (LBs), no enzyme has been identified for the initial C-terminal cleavage step that precedes multivesicular body (MVB)–associated maturation. This study aimed to define trafficking and post-translational processing differences between wild-type and disease-associated SP-C mutants using doxycycline-inducible mouse lung epithelial (MLE-12) cell lines expressing either SP-CWT or the common clinical variant SP-CI73T, validated using primary AT2 cells isolated from a murine SP-CI73T pulmonary fibrosis model and induced pluripotent stem cell (iPSC)-derived human alveolar type 2 cells (iATs) expressing the same mutant. Immunogold electron microscopy of SP-CWT mouse lung revealed robust WT proSP-C labeling in the Golgi, MVBs, & LBs consistent with normal anterograde trafficking. In contrast, SP-CI73T localized not only to the Golgi and MVBs but also to apical tubular structures beneath the plasma membrane, extending along the microvilli of the AT2 cell surface. Surface deposition of the mutant was confirmed by immunofluorescence in human I73T explant tissue and iAT2s, and by proteinase K protease protection assays showing extracellular exposure of proSP-CI73T, but not SP-CWT. To define/compare the routing pathways for WT and mutant SP-C, we used secretory pathway perturbations. Brefeldin A blocked processing of both isoforms, whereas a 20°C temperature shift selectively accumulated a processed SP-CWT intermediate, suggesting that its initial C-terminal cleavage occurs in the late-Golgi/trans-Golgi network (TGN). Subcellular fractionation of GFP-SP-CWT MLE-12 cells enriched both a full length proSP-C isoform and an intermediate bearing a partially cleaved COOH-propeptide. However, no proSP-C processing intermediates were identified in ER/Golgi fractions from GFP-SP-CI73T cells. SP-CWT processing was inhibited by Decanoyl-RVKR-CMK, a blocker of furin-related subtilisin-like proprotein convertases (PPCs). Mutating canonical PPC recognition residues (K160/R167) eliminated C-terminal processing, and overexpression of inhibitory pre-proprotein fragments of Furin or PC7—PPCs localized to the TGN—similarly prevented SP-CWT cleavage. By transmission electron microscopy the Golgi of AT2 cells from WT SFTPC mice appeared as organized stacks of flattened cisternae with closely apposed membranes; in contrast, SP-CI73T expressing AT2 cells exhibited a fragmented Golgi morphology, characterized by dispersed, swollen cisternae and unstacking. In conclusion, we have identified a previously uncharacterized early proteolytic processing step for SP-CWT shown to be essential to exclude aberrant transport of the proprotein to the plasma membrane, refining the model of SP-C trafficking, and highlighting the intricate regulation of protein processing in the Golgi. This more granular understanding of SP-C and lung epithelial biology will open new avenues for linking AT2 cargo defects and cell quality control dysfunction to diseases such as Idiopathic Pulmonary Fibrosis. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.