Cyr61 displays high-affinity binding to immobilized VTNC.

<p>(<b>A</b>) Sensorgrams for Cyr61 (in nM: <i>a</i>, 40; <i>b</i>, 20; <i>c</i>, 5; <i>d</i>, 2.5) binding to immobilized monomeric VTNC. (<b>B</b>) Sensorgrams show Cyr61 (in nM: <i>a</i>, 50; <i>b</i>, 25, <i>c</i>, 12.5; <i>d</i>, 6.25) binding to multimeric VTNC. Data were fitted using global two-state binding model. RU, resonance units. (<b>C</b>) and (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#s4" target="_blank">Materials and Methods</a>. (<b>E</b>) and (<b>F</b>) Semi log-transformation of the data depicted in (<b>C</b>) and (<b>D</b>), respectively. The (apparent) <i>K_D</i> 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).</p

Cyr61 prevents U937 cell adhesion to vitronectin.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#s4" target="_blank">Materials and Methods</a>. 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.</p

Kinetics of Cyr61 interactions with VTNC and SMTB ^1–44.

<p>Responses were obtained by injecting Cyr61 over immobilized VTNC or SMTB ^1–44 for 90 seconds at a flow rate of 30 µl/minute. Data fitting using two-site binding model (conformational change). Kinetic values obtained from the sensorgrams are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#pone-0009356-g002" target="_blank">Figure 2, C and D</a>, and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#pone-0009356-g003" target="_blank">Figure 3E</a>.</p

Kinetics of PAI-1 interactions with SMTB ^1–44.

<p>Responses were obtained by injecting stable mutant PAI-1 over immobilized SMTB ^1–44 for 90 seconds at a flow rate of 30 µl/minute. Data were derived from <i>Ka1</i> and <i>Kd1</i> and fitted using the Langmuir (1∶1 binding) equation. Kinetic values obtained from the sensorgrams presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#pone-0009356-g003" target="_blank">Figure 3D</a>.</p

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

<p>(<b>A</b>) SMTB ^1–44 was chemically synthesized and purified by reverse-phase chromatography. (<b>B</b>) Mass spectrometry for the synthetic peptide shows the expected mass for SMTB ^1–44. (<b>C</b>) Gel-filtration chromatography shows complex formation between PAI-1 and SMTB ^1–44 was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#s4" target="_blank">Materials and Methods</a>. (<b>D</b>) SPR experiments. Sensorgrams shows PAI-1 (in nM: <i>a</i>, 1.8; <i>b</i>, 0.9; <i>c</i>, 0.45; <i>d</i>, 0.225; <i>e</i>, 0.11) binding to immobilized SMTB ^1–44 domain. (<b>E</b>) Sensorgrams show Cyr61 (in nM: <i>a</i>, 12.5; <i>b</i>, 6.25; <i>c</i>, 3.1) binding to the SMTB ^1–44 domain. Data were fitted using global two-state binding model. RU, resonance units. (<b>F</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#s4" target="_blank">Materials and Methods</a>. (<b>G</b>) Semi-log transformation of the results presented in (<b>F</b>). The (apparent) <i>K_D</i> values for Cyr61/ SMTB ^1–44 interactions were calculated by nonlinear regression analysis of the binding data according to the Langmuir isotherm equation. (<b>H</b>) 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 (<b>E</b>). Experiments were performed in quadruplicate or quintuplicate (n = 3).</p

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

<p>(<b>A</b>) 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>B</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: Δ<i>H</i> = −24±1.34 kcal/mol. (<b>C</b>) Base line-adjusted heats per injection of SMTB ^1–44 (10 µM) into Cyr61 (1.0 µM). (<b>D</b>) Molar enthalpies per injection for SMTB ^1–44 interaction with Cyr61 where no heat exchange is observable. Filled circles, measured enthalpies.</p

Specificity of <i>An. gambiae</i> SGH hemagglutinating activity.

<p>*Inhibitory concentration; minimal concentration promoting inhibition of agglutination.</p>#<p>NI; non-inhibitory at the maximum tested concentration.</p><p>Specificity of <i>An. gambiae</i> SGH hemagglutinating activity.</p

Cyr61 binds to VTNC.

<p>(<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#pone.0009356-Holbourn1" target="_blank">[3]</a>. (<b>B</b>) Clustal alignment of CCN family members with IGFBP-3 and -5 indicates several conserved residues (*). (<b>C</b>) and (<b>D</b>) 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. (<b>E</b>) 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. (<b>F</b>) 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. (<b>G</b>) Cyr61 does not inhibit enzyme activity. Cyr61 (300 nM) was incubated with different enzymes and fluorogenic substrate hydrolysis was followed as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009356#s4" target="_blank">Materials and Methods</a>. SEM are not depicted but were less than 5% (n = 3).</p

Lectin activity for the SGH of Anophelinae mosquitoes, but not for other genus.

<p>(A) SGH from <i>An. gambiae</i> (male and female), <i>An. stephensi, An. freeborni, An. dirus, An. albimanus, Ae. aegypti, Culex quinquefasciatus, Phlebotomus duboscqi, Lutzomyia longipalpis,</i> were serially diluted followed by addition of O-type RBC. Two preparations of female <i>An. gambiae</i> SGH were tested. (B) Quantification of lectin activity. U/assay was given as 1/<i>n</i>, where <i>n</i> is the maximum dilution promoting agglutination presented in (A) ND, not detected. (C) Species-specificity. SGH of <i>An. gambiae</i> was tested in RBC from human, horse, sheep, goat, pig, cow, rat guinea pig, and chicken.</p

Expression of Agalectin, a member of the galectin family of lectin.

<p>(A) Clustal aligment of Agalectin with orthologs from Diptera, human galectin-1 and galectin-2. (B) Galectin purification. The chromatogram of the last purification step is shown. (C) SDS-PAGE of Agalectin, under reducing, or denaturing conditions. (D) Molecular weight determination. The retention volume of Agalectin was compared with molecular weight markers, and determined to behave as a dimer in solution. (E) Molecular modeling of Agalectin. Based on the galectin domain of porcine adenovirus (PDB 2WSU). (F) Lectin activity. Agalectin does not agglutinate RBC, with or without CaCl₂ (n = 3).</p