Degradation of collagen I by activated C1s in periodontal Ehlers-Danlos Syndrome

Abstract

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.

Keywords: C1r protease; Ehlers-Danlos Syndrome; collagen I; complement activation; periodontites.

Figure 1

Western blot analysis of collagen I expression in control (Co) and pEDS fibroblasts. Cell extracts and cell supernatants were probed with an anti-collagen I antibody. The signal at approx. 150 kDa represents procollagen, which was present in all fibroblasts; absence of this signal in the supernatant of control 1 may be due to rapid removal of the C-terminal antigen region for the collagen I antibody. Additional collagen I bands at approximately 72 kDa, 55 kDa and 43 kDa in pEDS fibroblasts and supernatants indicate cleavage of a proportion of collagen I. Differences in collagen I fragmentation may be attributed to the fact that the cells come from different donors and carry different C1R variants, as indicated. Expression of alpha tubulin was analysed as loading control. This figure is representative of three independent experiments with similar results.

Figure 2

Immunofluorescence analysis of collagen I in control (Co) and pEDS fibroblasts. Control fibroblasts exhibit a high abundant collagen network with strong fiber-like staining in the extracellular space. In patient fibroblasts, the quantity of the collagen network is strongly reduced and the fiber-like staining is weaker and thinner. This is a representative figure of four independent experiments. Scale = 20 µm.

Figure 3

Fluorescence images showing F-CHP staining on human control (Co) and pEDS fibroblasts. Fixed cells were stained using a fluorescence-labeled collagen-hybridizing peptide (CHP) which specifically binds to denatured or fragmented collagen fibers. In controls, F-CHP signals were weak indicating slow collagen I degradation. In patient fibroblasts, F-CHP was strongly increased with thick fiber-like structures on the cell surface indicating large amounts of denatured collagen I filaments. This figure is representative of four independent experiments. Scale = 20 µm.

Figure 4

Western blot analysis of C1s and collagen I in human fibroblasts with and without C1S knock-down. (A) Transfection of the same control fibroblast with different C1S-directed siRNAs identifies siRNA3 as the one that causes the strongest reduction of C1s expression. (B) siRNA3 transfection of pEDS fibroblasts leads to marked reduction of 72 kDa and 55 kDa collagen fragments, confirming C1s as cause for collagen I digestion in patient cells. Data are representative of three independent experiments.

Figure 5

Proteolytic activity of aC1s on procollagen I (A) Overnight incubation of collagen I from different sources with aC1s shows partial degradation into different smaller fragments as indicated by arrows. The degradation was independent from the source of collagen I Samples incubated without aC1s remained unaffected, as shown by the α1,α2, ß and γ collagen I chains. aC1s was represented by two bands, corresponding to the N-terminal chain at approx. 65 kDa and the C-terminal chain at approx. 32 kDa. (B) Denaturation of collagen I prior to incubation with aC1s shows almost complete degradation of procollagen I fibers into several smaller fragments. Samples incubated without aC1s remain unchanged. (C) C1s-mediated degradation of collagen I is temperature-dependent, with no degradation at an incubation temperature of 33°C, limited degradation at 37°C, and almost complete degradation at 40°C. These data suggest that the collagenase activity of aC1s depends on the unfolded state of collagen I (D) C1s collagenase activity is abolished by addition of the serine protease inhibitor PMSF at 10mM concentration, but lower PMSF concentrations have no inhibitory effect. The data are representative of five independent experiments.