b)

SUPPLEMENTARY FIGURE 1:A)FH1-5 mediated accelerated decay of the AP C3 convertase was measured by surface Plasmon resonance essentially as described in Schmidt et al, 2011. C3b (Complement Technology) was immobilised on an CM5 Chip (GE Healthcare) by amine coupling until about 130 resonance units were achieved. A mixture of FB (0.5 μM) and FD (60 nM) was applied for 120 s at 10 μl/min. The C3 convertase was allowed to decay for 210 s before injection of FH1-5 or FH1-4, (0.17 μM for 60 s). Both FH1-5 and FH1-4 (generated in Pichia Pastoris, see Wong et al, 2014) resulted in marked acceleration of convertase decay with FH1-5 showing better decay acceleration than FH1-4 as indicated by lower final response units. B)Decay acceleration function of FH1-5 was assayed using sheep erythrocytes pre-coated with AP convertase (see methods). Increasing concentration of FH1-5 or FH1-4 was added to FH depleted serum (and after adding FB) lysis was measured by detecting hemoglobin release at 420 nm (A420). Controls included 0% lysis (Buffer only - PBS/20 mM EDTA) and 100% lysis (0.01% Triton in PBS/20mM EDTA). Percentage of inhibition from lysis was calculated by the formula (A420[buffer only]-A420[FH])/A420[buffer only]*100%. Representative of 3 similar experiments.

a)

A.

0.04-0.5 μM

- ve

C3b α

FHFH1-5FH1-5^18-20

- ve

FH

FH1-5

FH1-5^18-20

0.04 -0.5μM

C3 α-chainC3b α-chain

C.

B.

SUPPLEMENTAL FIGURE 2:

(A) Fluid-phase FI cofactor activity of FH1-5^18-20 was measured by incubation of C3b and FI with for 30 min at 37 °C. Coomassie blue staining was used to visualise cleavage of the C3b α-chain in the reaction mixtures. For the negative control 3 μg of C3b was mixed with 0.045 μg FI in a total reaction volume of 20 μl. Equimolar concentrations of the recombinant proteins and full length FH were added ranging from 0.02-0.5 μM. The reactions were incubated for 20 min at 37 °C, mixed with 2x reducing SDS-PAGE sample buffer and analysed by SDS-PAGE on 10% gels followed by Coomassie blue staining of the gels. (B) C3b alpha chain band intensity densitometry of fluid phase assays, % Cleaved C3b α is shown from triplicate analysis, ± SD; FH to FH1-5^18-20 NS using two-way ANOVA with Bonferroni Multiple Comparisons Test (C) The effect of the recombinant FH proteins on the activity of the AP C3 convertase was tested in a fluid-phase assay as described by Wiesmann, et al, 2006. Nature 444: 217-220. Fluid-phase anti-C3 convertase activity of FH1-5^18-20 was measured by incubating C3, FB and FD in the presence of varying concentrations of FH, FH1-5 or FH1-5^18-20. To visualise C3 fragments, reactions mixtures were electrophoresed on 5% SDS-PAGE gels followed by immunoblotting (goat anti-human C3 polyserum and HRP-conjugated donkey anti-goat Ig secondary). In the absence of a regulator, C3 convertases form spontaneously and cleave C3 into C3a (8 kDa) and C3b as indicated by the presence of the lower molecular weight C3b α-chain in addition to the C3 α-chain in the negative control. With increasing concentrations of a regulator less C3b α-chain is present. Data is representative of several experiments.

A.

150

FH1-5

PBS

FH1-5^18-20

Glo

mer

ular

C3

(AFU

)

****100

50

0

SUPPLEMENTAL FIGURE 3: Effect of repeat administration of FH1-5^18-20 on glomerular staining of 8 week old, sex matched , FH-/- mice over 24 hours. (A) Glomerular C3 intensity was significantly reduced at the 24 hour timepoint compared to PBS and FH1-5. Horizontal bars denote median value; one-way ANOVA with Bonferroni Multiple Comparisons Test p<0.0001 compared to PBS and FH1-5. (B) Representative images of glomerular C3 (upper panel) and C3d (lower panel) at 24 hours. Scale Bar applicable to all images.

mC3

mC3d

FH1-5

PBS FH1-5^18-20

B.

50µm

SUPPLEMENTARY FIGURE 4:Six C57Bl/6 (wild type) mice were injected I.P. with a 3nmol dose of FH1-5^18-20 while two mice received only PBS as control. Remaining FH1-5^18-20 was detected in collected mouse serum (2, 6 and 24hr post injection) using the OX24 sandwich ELISA.

Supplemental Methods:

ELISA:Mouse C3 levels were measured by ELISA using HRP-conjugated goat polyclonal anti-mouse C3 Ab (MP Biomedicals, USA, Catalog N° 0855557) as previously described (Pickering et al, 2002). Alternatively, to detect human FH or our constructs, OX-24 was coated overnight at 5μg/ml on the ELISA plates. After washing with 200µL/well of 0.1% v/v TWEEN® 20 Detergent (Calbiochem, Germany) in PBS (PBST pH 7.2), plates were incubated with 100µL/well of 2% w/v BSA (Sigma-Aldrich, USA) in PBST for 1h at RT. After washing twice, plates were incubated with 50µL/well of each serum specimen diluted 1 in 12,000 for C3 detection or 1/1000 for FH detection in 2% BSA/PBST for 1h at RT. For the C3 standard curve, murine serum amyloid P component (Calbiochem, Germany), which contains mouse C3 protein at a concentration of 263mg/mL, was used in doubling dilutions commencing at 1 in 500. For the FH standard curve, Comptech FH was doubly diluted from 10μg/ml. After washing five times, plates were incubated with 50µL/well of 1 in 25,0000 HRP-conjugated goat anti-mouse C3 Ab or 1/4000 dilution of goat anti-human FH in PBST for 1h at RT. For FH reagent detection, plates were washed and probed with a 1/4000 dilution of bovine anti-goat-HRP in PBST for 1h at RT. After washing five times, plates were incubated with 50µL/well of TMB substrate reagents (BD, USA), after which the reaction was stopped using 20µL/well of 2N sulphuric acid (BD, USA). Spectrophotometry was performed at 450nm and when the optical density for the standard was plotted against the dilution factor on a logarithmic scale, a sigmoid curve was obtained. C3 or FH reagent concentration of mouse plasma samples was calculated from this standard curve using linear regression.

Sheep red blood cell (SRBC) based haemolytic assaysA FH loss of function assay was undertaken essentially as previously described by Sanchez-Corral et al, 2004, using normal human serum (NHS, negative control) and serum from an affected aHUS patient known to carry the CFH/CFHR1 hybrid gene (positive control, Venables et al, 2006). Sheep red blood cells (TCS Biologicals, UK) were washed several times in PBS and subsequently transferred to AP buffer (5 mM sodium barbitone [pH 7.4], 150 mM NaCl, 7 mM MgCl2, and 10 mM EGTA) for 2 further washes. Cells were resuspended at 0.1% and 100 µL plated out on round-bottomed 96-well plates containing 100 µL of triplicate serial dilutions of serum containing increasing quantities of FH reagents. Normal human serum was also pre-mixed with increasing quantity of OX-24 (1.2mg/ml in AP buffer) as needed. A duplicate serum dilution was set up in alternative pathway buffer plus 50mM EDTA to act as blank. Plates were incubated at 37°C for 30 minutes. To determine the amount of lysis, cells were pelleted by centrifugation, and hemoglobin release was measured at 420 nm (A420). Percentage of lysis was calculated by the formula ((A420[Sample]) - A420[buffer only])/ A420[100% lysis control])*100%.