Fig 1: High-confidence interaction among extracellular matrix (ECM)–related proteins and greater fibrosis in adult dilated cardiomyopathy (DCM) hearts compared with pediatric DCM hearts. A, STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis shows high-confidence interaction clusters (k-means=5 clusters indicated by node color) formed by ECM-related proteins. B through D, Representative images of Picrosirius Red (PSR) (B) and Masson trichrome–stained (C) left ventricular free wall from pediatric and adult control and DCM myocardium. Scale bar: 100 µm. D, Averaged quantification of collagen content (from PSR-stained sections) in indicated groups (mean±SEM). n=6 hearts per group. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. ADAM indicates a disintegrin and metalloprotease; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; AFU, arbitrary fluorescence units; BGN, biglycan; COL, collagen; DCN, decorin; LUM, lumican; MMP, matrix metalloprotease; NFC, nonfailing control; OGN, osteoglycan; SGCG, sarcoglycan gamma; SPARC, secreted protein acidic and cysteine-rich; THBS1, thrombosondin-1; TIMP, tissue inhibitors of metalloprotease; TNXB, tenascin x; VCAN, versican.
Fig 2: Cleavage of V1-5GAG by ADAMTS-4 and -5 deletion forms. (a) A schematic of the different ADAMTS-4 and -5 deletion variants used. (b) V1-5GAG (100 nM) was incubated with different variants of ADAMTS-5 and -4 (5 nM) for 2 h at 37 °C. Samples were then deglycosylated, subjected to SDS-PAGE and blotted either with the anti-Vc or anti-DPEAAE antibodies. Full-length anti-DPEAAE blot is presented in Supplementary Fig. 2.VSK, versikine. IB: immunoblot. (c,d) Time course experiments for cleavage of 50 nM V1-5GAG by ADAMTS-4 (5.5 nM; C) and -5 (0.2 nM; D) variants. The solid lines represent a nonlinear regression fit of the data as described in the Experimental procedures. (e) Michaelis-Menten curves for proteolysis of V1-5GAG by ADAMTS-5 MDTC. The enzyme (13 nM) was incubated with increasing concentrations of substrate. At the indicated time points, an aliquot was taken, stopped with EDTA and cleavage products measured by ELISA. Data are plotted as turnover number versus substrate concentration and are presented as mean ± SEM (n = 3–6).
Fig 3: Comparison of the versicanase activity of ADAMTS-1, -4 and -5. (a) Domain structures of V1, V1-5GAG and versikine (VSK), the N-terminal cleavage product resulting from ADAMTS-mediated cleavage at Glu441↓442Ala. (b) Coomassie staining of purified V1 and V1-5GAG following chondroitinase ABC digestion. Full-length gels are shown in Supplementary Fig. 1. (c,d) Versicanase activity of ADAMTS-1, -4 and -5. V1 (c) and V1-5GAG (d) (each 100 nM) were incubated with full length ADAMTS-1, -4 and -5 for 2 h at 37 °C. Samples were deglycosylated, subjected to SDS-PAGE and blotted either with polyclonal anti-Vc or anti-DPEAAE neoepitope antibodies. Enzyme concentrations were chosen with consideration for the relative difference in versicanase activity. IB: immunoblot. Full-length anti-DPEAAE blots are presented in Supplementary Fig. 1.
Fig 4: Kinetic constants for the versicanase activity of ADAMTS-4 and -5. (a) Representative ELISA standard curves of V1 and V1-5GAG digested with ADAMTS-5 (ATS-5). Following complete digestion of either V1 or V1-5GAG (100 nM) with ADAMTS-5, samples were diluted and incubated on a plate coated with anti-DPEAAE antibodies as reported in the Method Section. (b,c) Michaelis-Menten curves for proteolysis of V1-5GAG by ADAMTS-4 (b) and -5 (c). The enzyme (5.5 nM ADAMTS-4, 0.2 nM ADAMTS-5) was incubated with increasing concentrations of substrate. At the indicated time points, an aliquot was taken, proteolysis stopped with EDTA and cleavage products measured by ELISA. Data are plotted as turnover number versus substrate concentration and are presented as mean ± SEM (n = 3–4).
Fig 5: Versicanase activity of ADAMTS-4/13 and -5/13 Sp domain loop chimeras. (a,b) Molecular model of the ADAMTS-4 Sp domain highlighting loops ß1-ß2, ß3-ß4 and ß9-ß10. Structures of the ADAMTS-4 MD and ADAMTS-13 TCS variants were used as templates. In (a) the spatial localisation of the loops relatively to the rest of the molecule is shown, whereas in (b) the loops are highlighted in a cartoon model of the isolated Sp domain. (c) Sequences of the ß1-ß2, ß3-ß4 and ß9-ß10 loops in wild-type (WT) ADAMTS-4, -5 and the chimeras. (d) V1-5GAG (100 nM) was incubated with different variants of ADAMTS-4 and -5 (5 nM) for 2 h at 37 °C. Samples were then deglycosylated, subjected to SDS-PAGE and blotted either with either anti-Vc or anti-DPEAAE antibodies. Full-length anti-DPEAAE blot is presented in Supplementary Fig. 3. VSK, versikine. IB: immunoblot. (e,f) Time course experiments for cleavage of 50 nM V1-5GAG by ADAMTS-4 (1 nM; e) and -5 (0.2 nM; f) Sp domain loop chimeras. Data are presented as mean ± SEM (n = 3–6). The solid lines represent a nonlinear regression fit of the data as described in the Experimental procedures.
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