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

## TL;DR

This study explores how a disease-linked mutation in surfactant protein C (SP-C) disrupts its normal maturation and trafficking in lung cells, leading to chronic lung disease.

## Contribution

The study reveals distinct trafficking and processing pathways for wild-type and mutant SP-C, identifying a furin-like enzyme's role in SP-C maturation.

## Key findings

- Wild-type SP-C accumulates in lysosomal-related organelles, while the I73T mutant accumulates on the plasma membrane.
- Initial cleavage of SP-C occurs in the late-Golgi/trans-Golgi network via a furin-like proprotein convertase.
- Mutations in the PPC recognition site block SP-C processing, confirming the enzyme's role in maturation.

## Abstract

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-CI73T, validated using primary AT2 cells isolated from a murine SP-CI73T pulmonary fibrosis model and induced pluripotent stem cell–derived human AT2 cells expressing the same mutant. In all three models SP-CWT was highly concentrated in acidic lysosomal-related organelles while SP-CI73T 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-CI73T trafficking from SP-CWT. The exclusion of SP-CI73T 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-CWT intermediate, suggesting initial C-terminal cleavage of proSP-CWT 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-CWT 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.