Figure 1
Figure 1. CFHR1 binds to C3b and cells and competes with CFH. (A) CFHR1 is composed of 5 SCRs domains. SCR1 and SCR2 show 42% and 34% sequence identity to SCR6 and SCR7 of factor H (CFH), respectively. The 3 C-terminal SCRs show 100%, 100%, and 98% sequence identity to SCR18, SCR19, and SCR20 of CFH, respectively, which comprise the C-terminal surface binding region. (B) Equimolar concentrations of CFHR1 (25 μg/mL) and CFH (75 μg/mL) bind to immobilized C3b. Data represent mean values ± SDs from 3 separate experiments. Control represents binding of antibodies to immobilized BSA. (C) Plasma-derived CFHR1 (red curve) binds to HUVECs. Cells were incubated in human plasma, and bound CFHR1 was visualized with specific mAb (JHD10) by flow cytometry. Control: cells were treated with the secondary antibody alone (black curve). (D) Immunofluorescence staining of CFHR1 (green fluorescence) in renal and retinal human tissue. CFHR1 is detected at the lining of renal (i) and ocular (iv) blood vessels including large arteries, afferent and efferent arterioles associated with glomeruli (i), or the choriocapillaries (iv) as well as the Bruch membrane (nuclear counterstain: propidium iodide; original magnification, ×100). Preabsorption of mAb JHD10 with CFHR1 blocks reactivity (ii,v). In contrast, preabsorption with CFH (iii,vi) does not affect reactivity and demonstrates specificity of signals in panels i and iv. Thus, CFHR1 is present at the surface of endothelial cells and at the Bruch membrane. Inlays represent magnifications. Autofluorescence of lipofuscin-containing cells appears yellow. (E) HUVECs were incubated with a combination of CFHR1 (100 μg/mL) and CFH (100 μg/mL). After addition of the appropriate secondary antibodies, bound proteins were identified by confocal laser scanning microscopy. CFHR1 binding was detected with the CFHR1-specific mAb JHD10 in combination with a secondary anti–mouse antibody labeled with Alexa 647 (red fluorescence; i) and binding of CFH with a polyclonal antiserum specific for the N-terminal domains of CFH (anti–SCR1-4) together with a secondary goat anti–rabbit antibody labeled with Alexa 488 (ii; green fluorescence). An overlay of subpanels i and ii (iii) reveals colocalization of the 2 regulators as indicated by the yellow signal. Binding of primary (JHD10) and secondary antibodies showed no signal (control; iv). All cells were stained with DAPI to visualize the cell nucleus (bar represents 20 μm). (F) CFHR1 competes with factor H for heparin binding and thus affects the regulatory activity at surfaces. Constant amounts of factor H (10 μg/mL) were bound to immobilized heparin, and CFHR1 was used at increasing concentrations (0.1-20 μg/mL) as competitor. After competition, C3b and factor I were added. After incubation for 30 minutes the supernatant was removed, separated by SDS-PAGE, and transferred to a membrane, and C3b and degradation fragments were visualized with C3 antiserum (top panel). The mobility of the α' and β chain as well as the degradation fragments are indicated. A densitometric analysis as determined by the ratio of the α' 43 band and the β chain is shown in the bottom panel. A representative experiment of 3 separate experiments is shown.

CFHR1 binds to C3b and cells and competes with CFH. (A) CFHR1 is composed of 5 SCRs domains. SCR1 and SCR2 show 42% and 34% sequence identity to SCR6 and SCR7 of factor H (CFH), respectively. The 3 C-terminal SCRs show 100%, 100%, and 98% sequence identity to SCR18, SCR19, and SCR20 of CFH, respectively, which comprise the C-terminal surface binding region. (B) Equimolar concentrations of CFHR1 (25 μg/mL) and CFH (75 μg/mL) bind to immobilized C3b. Data represent mean values ± SDs from 3 separate experiments. Control represents binding of antibodies to immobilized BSA. (C) Plasma-derived CFHR1 (red curve) binds to HUVECs. Cells were incubated in human plasma, and bound CFHR1 was visualized with specific mAb (JHD10) by flow cytometry. Control: cells were treated with the secondary antibody alone (black curve). (D) Immunofluorescence staining of CFHR1 (green fluorescence) in renal and retinal human tissue. CFHR1 is detected at the lining of renal (i) and ocular (iv) blood vessels including large arteries, afferent and efferent arterioles associated with glomeruli (i), or the choriocapillaries (iv) as well as the Bruch membrane (nuclear counterstain: propidium iodide; original magnification, ×100). Preabsorption of mAb JHD10 with CFHR1 blocks reactivity (ii,v). In contrast, preabsorption with CFH (iii,vi) does not affect reactivity and demonstrates specificity of signals in panels i and iv. Thus, CFHR1 is present at the surface of endothelial cells and at the Bruch membrane. Inlays represent magnifications. Autofluorescence of lipofuscin-containing cells appears yellow. (E) HUVECs were incubated with a combination of CFHR1 (100 μg/mL) and CFH (100 μg/mL). After addition of the appropriate secondary antibodies, bound proteins were identified by confocal laser scanning microscopy. CFHR1 binding was detected with the CFHR1-specific mAb JHD10 in combination with a secondary anti–mouse antibody labeled with Alexa 647 (red fluorescence; i) and binding of CFH with a polyclonal antiserum specific for the N-terminal domains of CFH (anti–SCR1-4) together with a secondary goat anti–rabbit antibody labeled with Alexa 488 (ii; green fluorescence). An overlay of subpanels i and ii (iii) reveals colocalization of the 2 regulators as indicated by the yellow signal. Binding of primary (JHD10) and secondary antibodies showed no signal (control; iv). All cells were stained with DAPI to visualize the cell nucleus (bar represents 20 μm). (F) CFHR1 competes with factor H for heparin binding and thus affects the regulatory activity at surfaces. Constant amounts of factor H (10 μg/mL) were bound to immobilized heparin, and CFHR1 was used at increasing concentrations (0.1-20 μg/mL) as competitor. After competition, C3b and factor I were added. After incubation for 30 minutes the supernatant was removed, separated by SDS-PAGE, and transferred to a membrane, and C3b and degradation fragments were visualized with C3 antiserum (top panel). The mobility of the α' and β chain as well as the degradation fragments are indicated. A densitometric analysis as determined by the ratio of the α' 43 band and the β chain is shown in the bottom panel. A representative experiment of 3 separate experiments is shown.

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