The Brichos Domain-containing C-terminal Part of Pro-surfactant Protein C Binds to an Unfolded Poly-Val Transmembrane Segment

Abstract

Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane ␣-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to ␤-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a ϳ100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in ␤-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind ␣-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated ␤-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents ␤-sheet aggregation and that mutations in CTproSP-C can interfere with this function.

Mutations linked to interstitial lung disease can abrogate anti-amyloid function of prosurfactant protein C

“…1) and analysed for Congo red inclusions and amounts of aggregated and soluble proSP-C L188Q . An increase in SDS-soluble proSP-C L188Q levels was observed (data not shown), and was more pronounced than previously found after transfection with CTC lacking KDEL [24], as expected since the KDEL tag holds CTC in the ER. More importantly, transfection of CTC inhibited formation of Congo red positive inclusions (Fig.…”

“…Recombinant CTC L188Q does not aggregate in vitro [24], which suggests that the proSP-C L188Q inclusions seen in HEK293 cells (Fig. 2) may be mediated by aggregation of region(s) outside CTC.…”

“…The generation and culture of HEK293 cell lines stably expressing human wild type proSP-C, proSP-C L188Q and proSP-C ΔExon4 were previously described [24,25]; proSP-C I73T was constructed in a similar manner. A construct encoding the signal peptide from proSP-B, CTC, and the ER retention signal KDEL (CTC-KDEL) was cloned into pcDNA3.1 (InVitrogen) and constructs encoding proSP-C, proSP-C L188Q , proSP-C(Leu), or proSP-C(Leu) L188Q (Fig.1) were cloned into pIRES2-EGFP (BD Bioscience).…”

“…All samples were heated at 100°C for 4 minutes prior to separation by electrophoresis on 10-16% tricine gels followed by electrophoretic transfer to polyvinylidene difluoride membranes (Pall Corporation). After blocking with 5% non-fat dry milk, the membranes were probed with rabbit polyclonal antibodies directed against the N-terminal [26] or the C-terminal domain of proSP-C [24]. Enhanced chemiluminescence (Millipore) was used to detect antibody binding.…”

“…CTC was expressed and purified as described recently [24]. Briefly, a fragment covering residues 59-197 of human pro-SP-C (Fig.…”