Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Binding of Scl proteins to Factor H protein family members. A, CFHR1, CFHR2, CFHR3, and CFHR4A, as well as Factor H and FHL-1, were immobilized, and binding of streptococcal proteins Scl1.6, Scl1.55, and Scl2.28 was followed using a Strep-Tag II-specific HRP-coupled antibody. Streptococcal proteins Scl1.6 and Scl1.55 bound to CFHR1 and Factor H but not to any other Factor H family protein. Scl2.28 bound to none of the tested proteins (***, p < 0.001). B, 10% normal human serum was added to immobilized Scl1.6, Scl1.55, or Scl2.28, and bound CFHR1 and Factor H derived from human plasma were detected by a monoclonal antibody specific for CFHR1 (JHD10) and by a polyclonal antiserum specific for Factor H SCR1–4. The human regulators bound to Scl1.6 and Scl1.55 but not to Scl2.28. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments; ***, p < 0.001).

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Strep-tag, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Dose-dependent binding of CFHR1 and Factor H to streptococcal Scl1.6 and Scl1.55. Purified CFHR1 (A) and Factor H (B) bound dose-dependently to immobilized Scl1.6 (black circles) and Scl1.55 (black squares). No binding was detected for Scl1.1 (white triangles) and Scl2.28 (white diamonds). Error bars indicate S.D. C, binding of CFHR1 and Factor H to immobilized Scl1.6 (black), Scl1.55 (gray), Scl1.1 (light gray), and Scl2.28 (black dotted/dashed dotted) was analyzed by surface plasmon resonance SPR. CFHR1 (solid lines) and Factor H (dashed lines) bound to Scl1.6 and Scl1.55 but not to Scl1.1 or to Scl2.28. A representative experiment of three is shown.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Purification, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: S. pyogenes strains used in this study

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Purification

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Binding of CFHR1 and Factor H, and of deletion fragments to Scl1.6 and Scl1.55. A, binding of Scl1.6, Scl1.55, and Scl2.28 to CFHR1 and to the two CFHR1 fragments, SCR1–2 and SCR3–5, was assayed. Scl1.6 and Scl1.55, but not Scl2.28, bound to CFHR1 and the C-terminal CFHR1 fragment SCR3–5. No Scl protein bound to the N-terminal CFHR1 fragment SCR1–2. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments). The insert shows the schematic domain structure of CFHR1 and that of the two fragments (***, p < 0.001). B, binding of CFHR1 as well as CFHR1 fragments to immobilized Scl1.6 (light gray) and Scl1.55 (dark gray) was assayed by SPR. CFHR1 (solid line) and the C-terminal fragment SCR3–5 (dashed-dotted line) bound to Scl1.6 and Scl1.55. The N-terminal CFHR1 SCR1–2 fragment (dotted line) did not bind to Scl1.6 and Scl1.55. C, binding of Scl1.6, Scl1.55, and Scl2.28 to Factor H, and the indicated immobilized Factor H fragments was assayed by ELISA. Scl1.6 and Scl1.55 bound to full-length Factor H as well as the C-terminal Factor H fragments SCR15–20 and SCR19–20. Scl2.28 did not bind to any Factor H fragment tested. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments). The insert shows the schematic domain structure of Factor H and that of the various fragments (***, p < 0.001). D, binding was also analyzed by SPR. Again, the C-terminal Factor H SCR15–20 (dashed line) bound to immobilized Scl1.6 (light gray) and Scl1.55 (dark gray). The Factor H SCR15–18 fragment (dotted line) did not bind.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Identification of a linear Scl1.6-binding motif in SCR4 of CFHR1. A, linear peptides representing SCR4–5 of CFHR1 (residues 202–330), each with a length of 13 amino acids and an overlap of 10 residues, were spotted onto a membrane, and binding of Scl1.6 (upper panel) and Scl2.28 (lower panel) was followed. Scl1.6 bound to five spots representing the core motif, 232SVEYQ236 (upper panel). The nonbinding Scl2.28 did not bind to any peptide (lower panel). SCR4 of CFHR1 and SCR19 of the Factor H protein share sequence identity, and thus the binding motif is also contained in SCR19 (1133SVEYQ1137) of Factor H. B, structure of SCR4 and SCR5 of CFHR1 (corresponding to SCR19 and SCR20 of Factor H). The residues that form the linear binding motif are shown in black and are surface-exposed (Protein Data Bank code: 2G7I).

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Ionic strength (NaCl) and heparin affect CFHR1 and Factor H binding to Scl1.6 and Scl1.55. A, the influence of ionic strength on CFHR1 binding to immobilized Scl1.6 and Scl1.55 was analyzed by increasing the NaCl concentration. CFHR1 bound to immobilized Scl proteins in the absence of NaCl, and NaCl reduced binding in a dose-dependent manner. B, similarly, NaCl affected the interaction of Factor H with Scl1.6 and Scl1.55. The arrows (A and B) indicate the physiological NaCl concentration. C, increasing concentrations of heparin dose-dependently affected the binding of CFHR1 to immobilized Scl1.6 and Scl1.55. D, similarly, heparin affected the binding of Factor H to immobilized streptococcal Scl1.6 and Scl1.55 proteins. Again, the effect was dose-dependent. The mean values derived from at least three separate experiments ± S.D. are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; versus 0 mm NaCl or 0 μg/ml heparin.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Concentration Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Mutations in SCR20 and also disease-associated sequence variants affect Factor H binding to Scl1.6 and Scl1.55. Factor H SCR8–20, as well as four fragments that include aHUS-associated mutations (R1210C, V1197A, R1215G, and W1183V), and Factor H SCR15–20, which has heparin-binding residues exchanged (HepG), were attached via an immobilized monoclonal antibody (equal immobilization was verified in a parallel approach). Binding of Scl1.6, Scl1.55, and Scl2.28 applied to the fluid phase was detected using a Strep-Tag II-specific HRP-coupled antibody. Scl1.6 and Scl1.55, but not Scl2.28, bound to the unmodified fragment Factor H SCR8–20 and to Factor H SCR15–20, but the streptococcal proteins did not bind to any of the mutant proteins. Scl1.6 bound weakly to the Factor H SCR8–20 R1210C mutant construct. The mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments, ***, p < 0.001 versus no mutated proteins). The insert shows the structural model of SCR19–20 of Factor H (Protein Data Bank code: 2G7I). The positions of the five exchanged amino acids are indicated for the HepG mutant (upper panel). The disease-associated amino acids that were exchanged individually in four different aHUS cases are shown in black (lower panel). The linear Scl-binding motif is marked in dark gray.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Sequencing, Binding Assay, Strep-tag, Mutagenesis, Construct

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: CFHR1 and Factor H bind to various S. pyogenes serotypes. A and B, binding of CFHR1, Factor H SCR15–20, or Factor H SCR15–18 to S. pyogenes M1-, M6-, M28- and M55-type, which express Scl1.1, Scl1.6, Scl2.28, and Scl1.55, respectively, was assayed by flow cytometry (A), and the MFI are shown (B). CFHR1 and Factor H SCR15–20 bound to the Scl1.55-expressing M55-type and to the Scl1.6-expressing M6-type bacteria. Both proteins also bound with weaker intensity to the M28-type streptococci, which express the non-CFHR1/C-terminal Factor H-binding Scl1.28 and Scl2.28 surface proteins. The two human proteins did not bind to M1-type streptococci. Factor H SCR15–18 did not bind to any of the tested streptococcal serotypes. One representative experiment of five independent experiments is shown. C, binding of CFHR1 and of Factor H fragments to immobilized S. pyogenes serotypes M1, M6, M28, and M55 was analyzed by whole-cell ELISA. Bound proteins were identified with a polyclonal antiserum that detects CFHR1 and Factor H. The C-terminal CFHR1 SCR3–5, but not the N-terminal CFHR1 SCR1–2 construct, bound to all four streptococcal serotypes, however with different intensities. Factor H construct SCR15–20 also bound, whereas construct SCR15–18 did not bind. The background intensities were subtracted to allow direct comparison of the four serotypes. The mean values derived from at least three separate experiments ± S.D. are shown (***, p < 0.001).

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Factor H binding to Scl proteins is influenced by CFHR1, CFHR1 competes with Factor H for binding, and CFHR1 affects cofactor activity of Scl-bound Factor H. A, Factor H bound to Scl1.6 and Scl1.55 (columns 1 and 2). CFHR1 competes with Factor H for binding, and increasing amounts of CFHR1 affected Factor H binding in a dose-dependent manner (columns 3–10). Bound Factor H was detected with a Factor H SCR1–4-specific antiserum. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments). ***, p < 0.001 versus column 1 and 2. B, Factor H bound to Scl1.6 and Scl1.55 retains complement regulatory activity. Factor H bound to the indicated Scl proteins; then C3b and Factor I was added, and after incubation the mixture was separated by SDS-PAGE and transferred to a membrane. Cofactor activity of bound Factor H was analyzed by assaying C3b cleavage products by Western blotting. C3b cleavage results in the appearance of the α′ 68-, α′ 43-, and α′ 41-kDa fragments for Factor H bound to Scl1.6 (lane 1) or Scl1.55 (lane 3). A control reaction with Factor H in the fluid phase was assayed in lane 7. A representative experiment of three is shown. C, CFHR1 affects Factor H-mediated complement control at the level of C3 convertase. Factor H, used at constant amounts together with increasing concentrations of CFHR1, bound to immobilized Scl1.6 or Scl1.55 at molar ratios of Factor H:CFHR1 1:0.5, 1:1, 1:2, 1:4, and 1:8 (lanes 3–7 and lanes 10–14). Following washing, C3b and Factor I were added, and after incubation the mixture was separated by SDS-PAGE and transferred to a membrane. Cofactor activity of bound Factor H was analyzed by assaying the appearance of C3b cleavage products by Western blotting using a polyclonal C3b antiserum. C3b cleavage is identified by the appearance of the α′ 68-, α′ 43-, and α′ 41-kDa fragments. CFHR1, by displacing Factor H, affects Factor H cofactor activity on the level of the C3 convertase. A representative experiment of three is shown.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Activity Assay, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: CFHR1 bound to Scl1.6 and Scl1.55 inhibits TCC formation. A, the functional relevance of CFHR1·Scl1.6 and CFHR1·Scl1.55 complexes was analyzed in a hemolysis assay with sheep erythrocytes. TCC formation on sheep erythrocytes was induced with C5b6, C7, C8, and C9 and assayed by following erythrocyte lysis. Lysis was inhibited by increasing amounts of CFHR1·Scl1.6 and CFHR1·Scl1.55 complexes. Scl1.6 alone had a minor effect on hemolysis that was not amplified by increasing amounts. The mean values ± S.D. of three independent experiments are shown. B, TCC deposition was analyzed by the addition of TCC compounds C5b6, C7, C8, and C9 to CFHR1 bound to immobilized Scl1.6 and Scl1.55. For the negative control sample (no TCC), no C9 was added. TCC was detected using a TCC-specific antiserum. CFHR1 bound to Scl1.6 and Scl1.55 significantly reduced TCC deposition, whereas bound Factor H did not affect the TCC. Scl2.28, which bound to neither CFHR1 nor Factor H, did not influence TCC deposition. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Functional Assay, Hemolysis Assay, Lysis, Amplification, Negative Control