Journal: Journal of immunology (Baltimore, Md. : 1950)
Article Title: FHR5 Binds to Laminins, Uses Separate C3b and Surface-Binding Sites, and Activates Complement on Malondialdehyde-Acetaldehyde Surfaces.
doi: 10.4049/jimmunol.1701641
Figure Lengend Snippet: FIGURE 5. FHR5 binds to MAA-BSA via the SCRs5-7. (A) MAA-BSA (10 mg/ml) was coated onto the surface of a microtiter plate. After blocking, FHR5 (50 nM) was added and bound FHR5 was detected with a mAb amyc. Sh-BSA was used as control. FHR5 bound to immobilized MAA-BSA. (B) MAA-BSA (10 mg/ml) was immobilized and the FHR5 fragments (50 nM) were added. Fragment FHR55–7 bound to immobilized MAA-BSA, but FHR51–2, FHR53–4, FHR58–9 did not bind. FHR5 and the fragments were immobilized on the same microtiter plate. Therefore, binding intensities in (A) and (B) can be directly compared. Results represent mean 6 SD of three independent experiments. **p , 0.01. (C) Binding of FHR5 and the fragments to MAA-BSA was evaluated in real time by surface plasmon resonance. MAA-BSA was immobilized to the flow cells of a CM5 sensor chip and FHR5 or the fragments (100 nM) were flowed onto the MAA-BSA–coupled chip. FHR5 bound to MAA-BSA and fragment FHR55–7 also bound. A representative result of three independent experiments is shown. (D) To confirm that FHR5 binds to heparin and MAA-BSA via the same region, heparin (300 mg/ml) was added to fragment FHR55–7 (200 nM). After incubation, the complexes or FHR55–7 alone were flowed onto the MAA-BSA–coupled chip. Preincubation with heparin reduced binding of FHR55–7 to the MAA surface. A representative result of two independent experiments is shown. (E) The binding affinity of MAA-BSA to FHR5 was measured by biolayer interferometry. His-tagged FHR5 was loaded onto Ni-NTA biosensors (240 s). After blocking, MAA-BSA at the indicated concentrations was added and association was measured (240 s). Upon removal, the dissociation was followed for 240 s. The KD constant was calculated with BLItz-software. MAA-BSA bound with high affinity to FHR5 (KD 20.47 nM). A representative result of three independent experiments is shown. (F) MAA-BSA (10 mg/ml) was immobilized onto the surface of a microtiter plate. After blocking, NHS or serum from C3G patient #638 (20%) was added to the wells and incubated overnight at 4˚C. Following washing, MAA-bound proteins were detached from the surface, separated by SDS-PAGE, and transferred to a membrane. FHR5 and FHR21–2–FHR5 were detected by FHR5 antiserum. Serum-derived FHR5 and the hybrid protein FHR21–2–FHR5 from patient #638 bound to the MAA surface. The sh-BSA–coated surface served as a control. A representative Western blot of at least three independent experiments is shown.
Article Snippet: Bound FHR5 was visualized by FHR5 polyclonal antiserum (AF3845; R&D) in combination with the corresponding secondary antiserum (Dako).
Techniques: Blocking Assay, Control, Binding Assay, SPR Assay, Incubation, Software, SDS Page, Membrane, Derivative Assay, Western Blot
![FIGURE 6. FHR5 competes with factor H for MAA epitope binding. (A) MAA-BSA (10 mg/ml) was immobilized onto the surface of a microtiter plate. After blocking, FHR5 at increasing concentrations was mixed with factor H (250 nM) and then added to immobilized MAA-BSA. Following incubation, factor H was detected with a mAb [M16 (11)]. FHR5 inhibited factor H binding to MAA-BSA. The effect was dose dependent. (B) MAA-BSA (10 mg/ml) was immobilized. After blocking, 20% hiHS or heat-inactivated serum from C3G patient #638 was added to the wells either alone or in the presence of FHR5 or FHR2 (20 mg/ml), respectively. Bound factor H was detected with a mAb [M16 (11)]. The addition of FHR5 (322 nM) decreased serum derived factor H binding to MAA-BSA. In contrast, the addition of FHR2 had no influence. Factor H binding from patient serum was significantly lower compared with NHS. Sh-BSA was used for control. Results represent mean 6 SD of three independent experiments. **p , 0.01, ***p , 0.001. (C) FHR5 reduces factor H cofactor activity attached to an MAA surface. MAA-BSA (10 mg/ml) was immobilized onto the surface of a microtiter plate. After blocking, FHR5 (320 nM) and/or factor H (322 nM) were added as indicated. Following incubation and washing, C3b (140 nM) and factor I (220 nM) were added to each well. Following incubation, supernatants were collected and separated by SDS-PAGE, and proteins were transferred to a membrane. C3b cleavage fragments were detected by C3 antiserum. In presence of FHR5, factor H cofactor activity on MAA-BSAwas decreased. A representative Western blot of at least three independent experiments is shown. (D) The intensity of the a’43/a’41 bands was quantified. Result represents mean 6 SD of three measurements. ***p , 0.001.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3359/pm29483359/pm29483359__page10_image1.jpg)
