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1.
FIGURE 4

FIGURE 4. From: The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells is Impaired in Atypical Hemolytic Uremic Syndrome.

Summary figure of relative affinities of rH19-20 wildtype and mutant proteins for C3b and heparin, and of complement functional cell-based assays. The salt concentration (mM NaCl) at which the rH19-20 wildtype or mutant proteins eluted from the heparin affinity column are graphed on the x-axis. The relative affinity of the mutants versus the wildtype (WT) protein for C3b is graphed on the y-axis using the formula: relative affinity = [(Kd of WT rH19-20) / (Kd of each rH19-20 mutant)]. The average of the Kd values obtained on the C3b-coated CM5 and SA sensor chips were used. The higher values represent higher affinity for each ligand, while lower values represent the opposite. The symbols that represent each mutant depend on their % of remaining wildtype activity in the cell-based assay that measured the ability of the mutants to inhibit fH binding to C3b-coated host-like cells (Fig. 5A). These values are in agreement with the results obtained in the EH lysis assay (Fig. 6).

Viviana P. Ferreira, et al. J Immunol. ;182(11):7009-7018.
2.
FIGURE 1

FIGURE 1. From: The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells is Impaired in Atypical Hemolytic Uremic Syndrome.

Summary of mutagenesis study mapped onto 3-D structure of fH CCP 19-20. A, Side-chain heavy atoms of disease-linked mutations (left-hand panel) or non-disease linked mutations are drawn (in various colors for clarity) on cartoon representations of fH CCP 19-20. *V1197A was made in the context of the double mutant - V1197A;S1191L. B, A surface representation of mutated residues is superimposed on the structure (orthogonal views are shown; left-hand view is identical to that in A) and color-coded (see key) according to the predominant impact of the mutation on ligand binding. Positive and negative effects were not discriminated - “Heparin only” implies that the effect on C3b-binding was small while “C3b only” implies a small effect on heparin binding. The cyan color indicating “small effect” refers to that of L1189F on C3b and heparin binding. L1189R had significantly increased binding to both C3b and heparin.

Viviana P. Ferreira, et al. J Immunol. ;182(11):7009-7018.
3.
FIGURE 6

FIGURE 6. From: The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells is Impaired in Atypical Hemolytic Uremic Syndrome.

Ability of rH19-20 mutants to inhibit fH-mediated protection of human erythrocytes from complement-mediated lysis. Lysis of human erythrocytes (EH) was measured by pre-incubating anti-CD59-treated EH (5 × 106) with 1 μM wildtype or mutant rH19-20, in the presence of 40% NHS. This concentration of wildtype rH19-20 is enough to achieve ~ 50% hemolysis (IC50) of anti-CD59-treated cells, due to inhibition (by rH19-20) of fH-mediated protection. The mixture was incubated for 20 min, at 37°C. To determine the extent of hemolysis induced by the mutants versus the wildtype rH19-20, 200 μl cold GVBE was added, the samples were centrifuged and the optical density of the supernatant was determined at 414 nm. The percentage of lysis is graphed by subtracting the A414 of the background lysis control, as well as the percentage of lysis induced by blocking CD59 alone (~20%), and dividing this value by the maximum lysis obtained with water. The non-significant (n.s.) differences between the ability of the wildtype rH19-20 versus each of the mutants to induce lysis of EH (p>0.05) are indicated. The results are representative of two separate experiments shown as means and standard deviations of triplicate observations.

Viviana P. Ferreira, et al. J Immunol. ;182(11):7009-7018.
4.
FIGURE 5

FIGURE 5. From: The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells is Impaired in Atypical Hemolytic Uremic Syndrome.

The rH19-20 mutants are impaired in their ability to bind to C3b-coated host-like erythrocyte surfaces. A, Binding of radiolabeled fH to C3b on ESC3b cells in the presence of unlabeled wildtype or mutant rH19-20. Cells bearing 34 × 1011 C3b (1 μg) were incubated with ~ 20 ng of radioiodinated human fH and 0 to 8 μM of non-labeled wildtype or mutant rH19-20, in 100 μl DGVB. After 20 min at 22°C, the bound and free radiolabel were separated by centrifugation of cells through 20% sucrose in DGVB. The results are graphed relative to the initial binding observed with labeled fH in the absence of the rH19-20 proteins. Each experiment was carried out with five proteins at a time, using wildtype rH19-20 as an internal control in each set of assays. The average (n=5) wildtype rH19-20 IC50 is shown and was 0.20 +/− 0.04. The percent of inhibitory activity remaining for each protein is shown in parenthesis and was calculated using the following formula: (IC50 wildtype rH19-20 / IC50 mutant rH19-20) × 100. B, Same as above, except the C3b-coated cells were incubated with radioiodinated rH19-20 instead. The average wildtype (n=3) rH19-20 IC50 was 0.04 +/− 0.01. The left and right-hand panels in A-B show the data obtained with the aHUS-associated mutants or the designed mutants, respectively.

Viviana P. Ferreira, et al. J Immunol. ;182(11):7009-7018.
5.
FIGURE 2

FIGURE 2. From: The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells is Impaired in Atypical Hemolytic Uremic Syndrome.

Affinities of wildtype and mutant rH19-20 for glycosaminoglycans. Variants of rH19-20 were eluted from a HiTrap heparin-affinity chromatography column and protein elution was monitored using absorbance at 280 nm (A). In (A), left panel, for clarity, only the elution profile of the aHUS-associated mutant with the highest (W1183R) and the lowest (R1182S) affinity for heparin are shown in comparison to wildtype rH19-20. The conductivity values at which the aHUS-associated mutants eluted are summarized in (B), left panel. In (A), right panel, only the elution profile of the designed mutant with the highest (E1172R) and the lowest (R1203S) affinity for heparin are shown in comparison to wildtype rH19-20. The conductivity values at which all the designed mutants eluted are shown in (B), right panel. C, GMSA results. For these assays, 2-aminoacridone-tagged heparin-derived hexasaccharides were combined with rH19-20 or its mutants to give 34 μM protein and 40 μM oligosaccharide in 12 μl PBS containing 5 mM EDTA. To these samples were added 0.5 μl of glycerol and a trace of phenol red before incubation (15 minutes, room temperature). Samples were then loaded on a 1% agarose gel in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. Electrophoresis was performed (120 mV, 5 min) in a horizontal agarose electrophoresis system, using an electrophoresis buffer (40 mM Tris/acetate, 1 mM EDTA, pH 7.4). Immediately thereafter, the fluorescent oligosaccharides were visualized. The left and right-hand panels show the data obtained with the aHUS-associated mutants or the designed mutants, respectively. All results are representative of two separate experiments.

Viviana P. Ferreira, et al. J Immunol. ;182(11):7009-7018.
6.
FIGURE 3

FIGURE 3. From: The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells is Impaired in Atypical Hemolytic Uremic Syndrome.

Binding affinity of the rH19-20 mutants for C3b. A, Kd values for the binding of rH19-20 to C3b were measured on a CM5 sensor chip using surface plasmon resonance. Assays were performed on a BIAcore 3000 instrument by immobilizing 800-2000 response units (RU) of human C3b to three independent flow cells of a CM5 chip, using amine coupling. The rH19-20 mutants or wildtype rH19-20 were injected at 0-12 μM over the flow cells. The maximum equilibrium binding, at each concentration, was plotted using the Grafit 5 program, which determined the apparent Kd by fitting the data to a single-site saturation curve, where the capacity was fixed (as a constant) to that of the average (n=12) wildtype rH19-20 capacity. The standard deviations correspond to the Kd values obtained when samples were injected over the three different C3b-coated flow cells. B, Same as panel A, except biotinylated C3b was used to coat a streptavidin sensor chip. The non-significant (n.s.) differences between the ability of the wildtype rH19-20 versus each of the mutants to bind to C3b (p>0.05) are indicated. C, Representative equilibrium C3b-biotin binding curves for rH19-20 wildtype as well as a high binding (L1189R) and a low binding (R1215G) mutant. D, Analysis of the ability of rH19-20 mutants to compete with radiolabeled wildtype rH19-20 for binding to C3b on zymosan particles. Zymosan particles bearing 1 μg C3b were incubated with ~20 ng 125I-rH19-20 (wildtype) in combination with the IC50 dose of unlabeled wildtype rH19-20 (1.5 μM) or with 1.5 μM of the rH19-20 mutants in 100 μl DGVB. After 20 min at 22°C, the bound and free radiolabel were separated by centrifugation of cells through 20% sucrose in DGVB. The average (n=4) percent of 125I-rH19-20 remaining bound to the zymosan-C3b (ZymC3b) and standard deviations are shown for each protein. The differences between the ability of the wildtype versus each of the mutants to bind to ZymC3b were all significant (p<0.05). The results shown are from one of two representative experiments.

Viviana P. Ferreira, et al. J Immunol. ;182(11):7009-7018.

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