In vivo N-Terminomics Highlights Novel Functions of ADAMTS2 and ADAMTS14 in Skin Collagen Matrix Building

Abstract

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2 and ADAMTS14 were originally known for their ability to cleave the aminopropeptides of fibrillar collagens. Previous work using N-terminomic approach (N-TAILS) in vitro led to the identification of new substrates, including some molecules involved in TGF-β signaling. Here, N-TAILS was used to investigate the substrates of these two enzymes in vivo, by comparing the N-terminomes of the skin of wild type mice, mice deficient in ADAMTS2, in ADAMTS14 and in both ADAMTS2 and ADAMTS14. This study identified 68 potential extracellular and cell surface proteins, with the majority of them being cleaved by both enzymes. These analyses comfort their role in collagen matrix organization and suggest their implication in inflammatory processes. Regarding fibrillar collagen, this study demonstrates that both ADAMTS2 and ADAMTS14 are involved in the processing of the aminopropeptide of alpha1 and alpha2 type V collagen. It also revealed the existence of several cleavage sites in the Col1 domain and in the C-propeptide of type I collagens. In addition to collagens and other extracellular proteins, two major components of the cell cytoskeleton, actin and vimentin, were also identified as potential substrates. The latter data were confirmed in vitro using purified enzymes and could potentially indicate other functions for ADAMTS2 and 14. This original investigation of mouse skin degradomes by N-terminomic highlights the essential role of ADAMTS2 and ADAMTS14 in collagen matrix synthesis and turnover, and gives clues to better understand their functions in skin pathophysiology. Data are available via ProteomeXchange with identifier PXD022179.

Keywords: ADAMTS; Ehlers-Danlos Syndrome (EDS); N-Terminomics; TAILS; collagen; degradomics.

FIGURE 1

N-terminomic analysis of ADAMTS2 and 14 in mouse skin. (A) Schematic view of the experimental method to assess the potential substrates of ADAMTS2 and/or ADAMTS14 in mouse skin and to investigate their biological function in vivo. Three independent iTRAQ-TAILS analyses using skin proteome of each genotypes (4-plex) have been performed. As a general procedure, a protease/control (P/C) ratio above or below the three sigma cutoff from the normal distribution of natural N-termini is considered to be related to the studied protease. Here, the ratio Wt/TS2^−/− and TS14^−/−/TS2^−/−TS14^−/− were used to investigate the ADAMTS2 substrates, the ratios Wt/TS14^−/− and TS2^−/−/TS2^−/−TS14^−/− were used to investigate the ADAMTS14 substrates and the ratio Wt/TS2^−/−TS14^−/− was used to investigate the substrates common to ADAMTS2 and ADAMTS14. (B) Venn diagram showing the number of specific or common total (left) or labeled (right) proteins identified in each experiment. Experiment A is in blue, B in green and C in purple. Venn diagrams were generated using the online BioVenn software (Hulsen et al., 2008).

FIGURE 2

ADAMTS2 and 14 degradomes in mouse skin and their biological processes. (A) Schematic view of the multiple comparisons of the P/C ratios analyzed for each iTRAQ-TAILS experiment (n = 3). The function of the ADAMTS2 (in blue) is investigated by the comparison of wild type (Wt) mice with those deficient for ADAMTS2 (TS2^−/−) and from the comparison of the mice deficient for ADAMTS14 (TS14^−/−) with those deficient for ADAMTS2 and ADAMTS14 (TS2^−/−TS14^−/−). The potential substrates of ADAMTS14 (in green) were identified from the comparison of Wt mice with the mice deficient for ADAMTS14 (TS14^−/−) and from the comparison of the mice deficient for ADAMTS2 (TS2^−/−) with those deficient for ADAMTS2 and ADAMTS14 (TS2^−/−TS14^−/−). The common substrates of ADAMTS2 and 14 (red) were obtained from the comparison between Wt mice and TS2^−/−TS14^−/− mice, and by combining data obtained specifically for Adamts2 and for Adamts14. (B) Venn diagram showing the common and specific N-terminally labeled proteins related to ADAMTS2 (blue), ADAMTS14 (green) or ADAMTS2 and ADAMTS14 (green). All the 437 unique proteins (287 identified by comparing Wt to TS2^−/−, 408 by comparing Wt to TS14^−/− and 278 by comparing Wt to TS2^−/−TS14^−/−) showing a P/C ratio above or below three sigma cutoff from the normal distribution of natural N-termini are reported on the left. The 68 extracellular and cell surface proteins related to ADAMTS2 or ADAMTS14 activities (45 by comparing Wt to TS2^−/−, 61 by comparing Wt to TS14^−/− and 50 by comparing Wt to TS2^−/−TS14^−/−) are reported on the right. (C) Biological processes related to all the proteins affected by ADAMTS2 and ADAMTS14 in mouse skin (left panel) or related to the extracellular and cell surface substrates (right panel). The biological processes have been identified using the Panther database (Mi et al., 2019). Statistical Overrepresentation test was performed using the whole Mus musculus as reference dataset, the pValue is determined using the Fisher’s Exact test and corrected by the determination of the false discovery rate (fdr). The fdr is below 0.05 for all the biological processes reported. nd: not detected.

FIGURE 3

ADAMTS2 and/or ADAMTS14 cleavages of type I and V collagens. Schematic view of the pro alpha-1(I) and alpha-2(I) chains showing the signal peptides (PS), the main triple helix collagenous domain (COL1) and the short triple helix collagenous domain (COL2) and the N- and C-propeptides (N- and C-Pro) (A). Schematic view of the pro alpha-1(V) and alpha-2(V) chains showing the collagenous (COL) and non collagenous (NC) domains (B). The ADAMTS2 and ADAMTS14 cleavage sites are indicated by arrows. The red arrows correspond to the already reported ADAMTS2 cleavage sites (Colige et al., 2005; Bekhouche and Colige, 2015). Table showing the P/C ratio for type V collagens, according to the cleavage sites observed by proteomic. Ratios above the average 3σ cut-offs are highlighted in green. The previously described ADAMTS2 cleavage site within alpha-1(V) chain is in bold (C).

FIGURE 4

Collagen digestion from calf skin by recombinant human ADAMTS2 and/or ADAMTS14 (A–F) Collagen from dermatospatactic calf (DC) skin has been heat denatured (10 min at 95°C) (B, D, F) or not (A, C, E) before addition of recombinant human TS2 and/or TS14 overnight at 37°C. Digestion products have been analyzed by 6.25% (A, B) or 10% (C–F) SDS-PAGE stained by Coomassie blue. Pictures at (E) and (F) correspond respectively to (C) and (D) with a higher contrast to emphasize the degradation trail. Collagen from normal calf skin (NC) have been used as control for the identification of the fully processed α1 and α2 chains. Products at 31 kDa (C, E) correspond to the N-terminal propeptide released mainly by ADAMTS2 when type I collagen is in its native form. Additional low MW products can be observed after incubation of denatured collagen with ADAMTS14 and ADAMTS2.

FIGURE 5

Cleavage of type I procollagen. The collagen substrate, mainly in the form of pro-α1I and pro-α2I, was recovered from culture medium conditioned by dermatosparactic fibroblasts, and then incubated with recombinant human ADAMTS2 and/or ADAMTS14 during 18 h at 37°C. Digestion products were analyzed by 6.25% SDS-PAGE stained by Coomassie blue. The cleavage of pro-α2 into pCα2 and mature α2 demonstrates that ADAMTS2 displays a procollagen C-proteinase activity in addition to its aminoprocollagen peptidase property.

FIGURE 6

In vitro cleavage of actin and vimentin by ADAMTS2 and ADAMTS14. Table showing the P/C ratios according to the cleavage sites observed by proteomic. Ratios above the average 3σ cut-offs are highlighted in green (A). Western blot analysis of in vitro cleavage of actin (B) and vimentin (C) by ADAMTS2 and/or ADAMTS14. The schematic representation of actin and vimentin are reported on the top of the western blots with their respective actinin binding (magenta) and coiled coil (blue) domains. Human fibroblasts lysates were incubated in the presence of recombinant ADAMTS2 and/or ADAMTS14 (200 nM, 16 h at 37°C). EDTA was used as an inhibitor of metalloproteinases. Degradation products are indicated by black arrows.

FIGURE 7

Cleavage site specificity of ADAMTS2 and ADAMTS14 in mouse skin. The cleavage site specificity of ADAMTS2 (left panels) or ADAMTS14 (right panels) was determined using all the extracellular or extracellular transmembrane cleavage sites (top), without type I collagen (middle panels) or without all the fibrillar collagens (down panels). The amino acid sequence logos, corrected by the natural abundance of amino acids in the human proteome, were generated using the iceLogo software package (Colaert et al., 2009). The height of the amino acids represents their frequency regarding their natural abundance; the color reflects its physicochemical properties.