TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

Abstract

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

Keywords: ADAMTS; ADAMTS7; Biomarkers; Coronary artery disease; Degradomics; EFEMP1; Mass spectrometry; Protease; Substrate identification; TAILS proteomics.

Graphical abstract

Fig. 1

ADAMTS7 autocleavage sites detected in TAILS experiments.A, ADAMTS7 protein domains and locations of the TAILS significantly regulated WT/EQ peptides indicating autocleavage events. B, ADAMTS7 autocleavage peptide total intensities pooled from all TAILS experiments to show the relative abundance of each event. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; Cys-rich, cysteine-rich; Dis, disintegrin; PL, protease and lacunin; T, thrombospondin repeats; TAILS, terminal amine isotopic labeling of substrates.

Fig. 2

Volcano plots of TAILS-regulated peptides visualizing the high confidence cleavage sites.A–C, comparison of WT/EQ- and WT/Luc-regulated peptides from three independent TAILS experiments after removal of mouse ADAMTS7 peptides. A, TAILS SMC1 (p < 0.01); B, TAILS SMC2 (p < 0.05); C, TAILS HUVEC (p < 0.05), dotted line reflects significance cut off on the -log10P scale. Regulated peptides enriched for ADAMTS7 WT activity and meeting all criterial for the high confidence candidate cleavage sites (significant for both WT/EQ and WT/Luc comparisons) are shown in green for each TAILS experiment. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; HUVEC, Human umbilical vein endothelial cells; SMC, smooth muscle cells; TAILS, terminal amine isotopic labeling of substrates.

Fig. 3

ADAMTS7 TAILS high confidence cleavage sites from independent experiments. Histograms showing the overlap between significantly regulated candidate cleavage sites from the SMC1 (A), SMC2 (C), and HUVEC (E) TAILS experiments. Candidate cleavage sites present in both the WT/EQ and WT/Luc comparisons were consistently associated with ADAMTS7 activity and are defined as high confidence cleavage sites (shown as green dots in Fig. 2). The remaining regulated peptides were significant for one or more condition(s) in the histogram categories. Analysis of the cleavage sites using iceLogo shows the similarities between independent TAILS experiments for SMC1 (B), SMC2 (D), and HUVEC (F). ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; HUVEC, Human umbilical vein endothelial cells; SMC, smooth muscle cells; TAILS, terminal amine isotopic labeling of substrates.

Fig. 4

ADAMTS7 TAILS Discovery Set Overlap Analysis.A, venn diagram showing the overlap of unique candidate cleavage sites from SMC1 and SMC2 high confidence sites. B, venn diagram showing the overlap from all SMC and HUVEC TAILS datasets. C, gene assignment of the 91 unique candidate cleavage sites identified from multiple TAILS experiments, including 24 unique sites from 16 different genes identified in all three TAILS datasets. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; HUVEC, Human umbilical vein endothelial cells; SMC, smooth muscle cell; TAILS, terminal amine isotopic labeling of substratess.

Fig. 5

Validation of TAILS substrate EFEMP1 and cleavage site preference.A, EFEMP1/Fibulin-3 protein domains, amino acid sequence of the atypical EGF repeat linker, and location of the ADAMTS7 cleavage sites. Abbreviated EFEMP1 domains: signal peptide (SP), N-terminal region (N), EGF repeats (E). B, concentrated medium from HUVEC expressing Ad-Luc, Ad-mWT, or Ad-mEQ assessed by Western blot under nonreducing conditions. Anti-EFEMP1 antibody recognizes an epitope C-terminal to the ADAMTS7 cleavage sites. C, quantitation of semi-tryptic or semi-chymotryptic peptides from HUVEC medium matching novel cleavage sites from the endogenous EFEMP1 protein. The total area was greater for the 123.124 cleavage site compared to the adjacent 124.125 cleavage site. Additional cleavage events observed were also found in the Luc and EQ controls. D, in vitro cleavage of HA-EFEMP1 by purified full-length mouse ADAMTS7 S3A assessed by Western blot. The antibodies to the N-terminal HA epitope and C-terminal EFEMP1 epitope recognized the EFEMP1 more strongly under nonreducing conditions. A band at 100 kDa under nonreducing conditions is consistent with a purified HA-EFEMP1 dimer, which was also sensitive to ADAMTS7 cleavage. E, overnight digest of HA-EFEMP1 by mouse ADAMTS7 S3A assessed by Coomassie staining. F, quantitation of semi-tryptic or semi-chymotryptic peptides from the atypical EGF1 repeat region from HA-EFEMP1, showing a consistent preference for the 123.124 cleavage site. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; E, EGF repeats; HUVEC, Human umbilical vein endothelial cells; N, N-terminal region; SP, signal peptides; TAILS, terminal amine isotopic labeling of substrates.