Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44) domain of vitronectin

Abstract

Background: Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV) family of extracellular-associated (matricellular) proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C (vWF), thrombospondin type 1 (TSP), and C-terminal growth factor cysteine knot (CT) domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.

Methods and findings: In this report, surface plasmon resonance (SPR) experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC) with K(D) in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44)) of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44), and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44) in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44) at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules.

Conclusions: The finding that Cyr61 interacts with the SMTB(1-44) domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

Figure 1. Cyr61 binds to VTNC.

(A) Structure of CCN family members. CCN1 (Cyr61, represented in the figure), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) have a shared structure consisting of a secretory signal peptide (SP), an IGFBP domain (Module I), a von Willebrand type C domain (vWC, Module II), a thrombospondin-1 domain (TSP, Module III) and a cysteine knot (CT, Module IV) domain. Domains are linked by hinge regions susceptible to protease cleavage. Modified from reference . (B) Clustal alignment of CCN family members with IGFBP-3 and -5 indicates several conserved residues (*). (C) and (D) SDS-PAGE for IGFBP-1 to -5, and Cyr61, respectively. Two µg of proteins were loaded in a 4–12% NU-PAGE gel, which was Coomassie blue stained. (E) SPR experiments: proteins (100 nM) were used as analytes for immobilized VTNC. Binding levels at stability was used as a value for comparison. RU, resonance units. (F) Specificity of Cyr61 (30 nM) tested by solid-phase binding assays: proteins were immobilized as the following densities/well: VTNC (25 ng), fibrilar collagen (50 ng), ixolaris (75 ng), soluble collagen and laminin (100 ng), followed by incubation with Cyr61 (30 nM). Bound-Cyr61 was identified using anti-Cyr61 monoclonal antibody. (G) Cyr61 does not inhibit enzyme activity. Cyr61 (300 nM) was incubated with different enzymes and fluorogenic substrate hydrolysis was followed as indicated in Materials and Methods. SEM are not depicted but were less than 5% (n = 3).

Figure 2. Cyr61 displays high-affinity binding to immobilized VTNC.

(A) Sensorgrams for Cyr61 (in nM: a, 40; b, 20; c, 5; d, 2.5) binding to immobilized monomeric VTNC. (B) Sensorgrams show Cyr61 (in nM: a, 50; b, 25, c, 12.5; d, 6.25) binding to multimeric VTNC. Data were fitted using global two-state binding model. RU, resonance units. (C) and (D) solid-phase-binding assay for Cyr61 (0–1 µM) interaction with immobilized monomeric or multimeric VTNC, respectively. Binding was estimated with anti-Cyr61 monoclonal antibody followed by alkaline-phosphatase labeled anti-mouse secondary antibody and appropriate substrate as described in Materials and Methods. (E) and (F) Semi log-transformation of the data depicted in (C) and (D), respectively. The (apparent) K_D values for Cyr61/VTNC interactions were calculated by nonlinear regression analysis of the binding data according to the Langmuir isotherm equation. All treatments were performed in quadruplicate or quintuplicate (n = 3).

Figure 3. Cyr61 displays high-affinity binding to SMTB ^1–-44 domain.

(A) SMTB ^1–44 was chemically synthesized and purified by reverse-phase chromatography. (B) Mass spectrometry for the synthetic peptide shows the expected mass for SMTB ^1–44. (C) Gel-filtration chromatography shows complex formation between PAI-1 and SMTB ^1–44 was performed as in Materials and Methods. (D) SPR experiments. Sensorgrams shows PAI-1 (in nM: a, 1.8; b, 0.9; c, 0.45; d, 0.225; e, 0.11) binding to immobilized SMTB ^1–44 domain. (E) Sensorgrams show Cyr61 (in nM: a, 12.5; b, 6.25; c, 3.1) binding to the SMTB ^1–44 domain. Data were fitted using global two-state binding model. RU, resonance units. (F) Solid-phase binding assay. Cyr61 (0–1 µM) was incubated with immobilized SMTB ^1–44 and binding estimated with anti-Cyr61 monoclonal antibody, followed by alkaline phosphatase-labeled anti-mouse secondary antibody and appropriate substrate as described in Materials and Methods. (G) Semi-log transformation of the results presented in (F). The (apparent) K_D values for Cyr61/ SMTB ^1–44 interactions were calculated by nonlinear regression analysis of the binding data according to the Langmuir isotherm equation. (H) Competition experiments. Cyr61 (30 nM) was incubated with 0, 50, and 500 nM PAI-1, or 500 nM ixolaris. Mixtures were added to SMTB ^1–44-coated wells and incubated for 90 minutes. SMTB ^1–44-bound Cyr61 was estimated as in (E). Experiments were performed in quadruplicate or quintuplicate (n = 3).

Figure 4. Cyr61 does not interact with SMTB ^1–44 domain in solution.

(A) Isothermal titration calorimetry for stable mutant PAI-1 interaction with SMTB ^1–44. Base line-adjusted heats per injection of SMTB ^1–44 (10 µM) into PAI-1 (1.0 µM). (B) Molar enthalpies per injection for PAI-1 interaction with SMTB ^1–44. Filled circles, measured enthalpies; solid line, fit of experimental data to a single-site binding model. Thermodynamic parameters: ΔH = −24±1.34 kcal/mol. (C) Base line-adjusted heats per injection of SMTB ^1–44 (10 µM) into Cyr61 (1.0 µM). (D) Molar enthalpies per injection for SMTB ^1–44 interaction with Cyr61 where no heat exchange is observable. Filled circles, measured enthalpies.

Figure 5. Competitive ELISA.

Indicated concentrations of native monomeric VTNC, heat-modified monomeric VTNC, multimeric VTNC, SMTB ^1–44, or ixolaris were co-incubated with a constant concentration of Cyr61 (30 nM) on microtiter wells coated with multimeric VTNC. The binding of Cyr61 to immobilized VTNC was detected with anti-Cyr monoclonal antibody followed by alkaline phosphatase-labeled anti-mouse secondary antibody and appropriate substrate as described in Materials and Methods. Results were from quadruplicate experiments.

Figure 6. Cyr61 prevents U937 cell adhesion to vitronectin.

uPAR-bearing U937 cells were incubated for 120 minutes with VTNC-coated wells previously incubated with buffer or Cyr61 or PAI-1 (1.5 µM) as described in Materials and Methods. As a control, wells were coated with gelatin only. One hundred % cell adhesion was estimated in the presence of buffer (vehicle) only, and 0% as adhesion to gelatin. Results from quintuplicate experiments are shown.