Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome
Blood Roumenina et al.
114: 2837
Supplemental materials for: Roumenina et al
Supplemental Methods
Genetic analysis
Genomic DNA from each patient was obtained from peripheral-blood leukocytes. Coding sequences of the FB gene were amplified with primers flanking all 18 exons (The primer sequences are available from the authors upon request), purified using the Multiscreen® plates (Millipore, Molsheim, France), and sequenced with 96 capillary Sequencer 3730 using the dye terminator method (Applied Biosystems, Courtaboeuf, France). Large genomic rearrangements like CFHR1/3 deletion were studied my multiplex ligand-dependent probe amplification (MLPA) as described.1
Structure analysis
The crystal structures of FB, Bb and von Willebrand type A (vWF-A) domain are available in Protein Data Bank (PDB ID 2OK5, 1RRK and 1Q05 respectively). Molecular graphic imaging and analysis were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (http://www.cgl.ucsf.edu/chimera/).2 Protein numbering throughout this study is according to the sequence of the mature protein, without signal peptide. Wherever appropriate, the numbering according to the gene sequence (starting with c.) and to the protein with leader peptide (starting p., having additional 25 residues) will be given for reference.
Recombinant FB production and mutagenesis
The mRNA was extracted from peripheral blood mononuclear cells from a healthy donor. Reverse transcription-PCR (RT-PCR) was performed (1st Strand cDNA Synthesis Kit for RT-PCR AMV+, Roche, Mannheim, Germany) and the complement FB cDNA was amplified by PCR with the High Fidelity DNA polymerase, (Roche, Mannheim, Germany) using a forward primer (5′ –TTC CAA CGC CAT GGG GAG- 3′) and a reverse primer (5′ –TCC AGC AGG AAA CCC CTT ATA G- 3′). It was cloned in the eukaryote expression vector pcDN3.1/V5-His TOPO® TA (Invitrogen, Paisley, Great Britain). The stop codon was introduced before the V5 and HisTag sequences, allowing synthesis only of a native sequence of FB. The D254G and K325N were introduced by site-directed mutagenesis using the QuikChange® II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s instructions. The constructs were completely sequenced to confirm that no additional mutations had been introduced. The transient expression of recombinant FB proteins was conducted in transitory transfected HEK-293T cells cultured in DMEM+glutaMAX™-I 4,5g/l D-Glucose + Pyruvate medium (Gibco, Paisley, GB) containing 10% foetal calf serum.
Recombinant FB characterization
The integrity of recombinant wild type (WT) and mutants FB was tested by Western blot. The proteins in the culture supernatants were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with biotinilated polyclonal sheep anti-human FB antibody (Abcam), followed by Streptavidin-HRP (Amersham) and ECL (GE Healthcare).
The FB content was assessed by sandwich ELISA, using immobilized polyclonal sheep anti-human FB antibody (Abcam) for capturing and biotinylated sheep anti-human FB, followed by streptavidin-Horseradish Peroxydase (HRP) (Amersham) for detection. The expression levels were similar between WT and mutant forms.
The recombinant proteins were purified by immunoafinity chromatography as described.3 The purity was accessed by SDS-PAGE and coomassie blue staining. The integrity of the purified proteins was accessed by Western blot as above.
ELISA for FB binding to C3(H2O) in presence of divalent ions and EDTA
Microtitre wells were coated with purified human C3(H2O) (Calbiochem, La Jolla, CA), at 0.5µg/well in PBS, pH 7.4 for 1 h at 37°C, any residual binding sites were blocked with 1% BSA for 1 h at 37°C and washed with PBS pH 7.4, containing 0.05% Tween 20. Serial dilutions of the recombinant proteins in a low ionic strength (75mM NaCl) 10mM HEPES buffer, containing 10mM Mg2+, 1mM Ni2+, or 5mM EDTA pH 7.4 were incubated for 45 min at 37°C. Following washing with the same HEPES buffer, wells were incubated with a biotinylated sheep polyclonal anti-human FB antibody (1:1000, Abcam) for 1 h at 37°C. The amount of bound recombinant protein was detected by incubation with Streptavidin-HRP (1:1000) conjugate. Colour was developed using OPD substrate and OD490 nm was measured. The background binding was subtracted.
Inhibitory ELISA – ability C3(H2O), C3b or iC3b to inhibit the interaction of recombinant FB proteins with C3(H2O), C3b, or iC3b
Native C3(H2O), C3b or iC3b were coated to the plate at 0.3 µg/well. The supernatants containing 0.3µg/well recombinant FB were mixed with increasing concentrations of the corresponding C3 form (0, 0.3, 0.6 µg/well) and applied to the wells. The binding of FB proteins was accessed as above.
Inhibitory ELISA — ability of recombinant FB proteins to inhibit the interaction of native FB with C3(H2O)
Native FB (CompTech, 0.3 µg/well) was coated to the ELISA plate. Serial dilutions of supernatants containing the same amount of the WT, D254G, K325N (starting from 1µg/well) or the maximal volume of the SN0 were pre-incubated with C3(H2O) (0.3 µg/well). After blocking (1% BSA) the supernatants containing C3(H2O) were applied to FB coated plate. Bound C3(H2O) was detected using biotinilated anti-C3 polyclonal antibody (kind gift from Prof. Marc Fontaine, Rouen , France), followed by streptavidin-HRP and TMB as a substrate.
Induction of apoptotsis ot adherent quiescent HUIVEC monolayers
Adherent, quiescent confluent HUVEC monolayers at passage 3 (12 wells plates) were washed with PBS and incubated with 400 nM staurosporine for 3h in M199 medium containing 10% FCS. Detached cells were collected, washed, and treated as described for the necrotic cells.
Supplemental Results
Integrity of recombinant FB proteins
The integrity of the recombinant FB proteins was demonstrated by a Western blot (Fig. S1). The recombinant WT, D254G and K325N were indistinguishable from the native FB, purified from plasma. The polyclonal antibody against FB did not recognize FB protein in the SN0, transfected with the vector only.
Specificity of the interaction between the recombinant FB and C3(H2O), C3b, and iC3b
The specificity of the interaction between recombinant FB proteins and different forms of C3 was accessed by an inhibitory ELISA. WT FB bound to C3(H2O) and C3b but not to iC3b (Fig. S2A). The two mutants had increased binding to C3(H2O) and C3b. They bound to iC3b in contrast to the WT. Addition of iC3b in the fluid phase inhibited these interactions (Fig. S2B). Increased concentrations of fluid phase C3(H2O) (Fig. S2C) inhibited interaction of the recombinant FB proteins with C3(H2O) coated to the plate. The same situation was observed for C3b (Fig. S2D).
Recombinant FB proteins compete with native FB for C3 binding
The interaction between native FB coated to the plate and C3(H2O) in fluid phase was specifically inhibited by addition of SN containing recombinant WT or mutant FB (Fig. S3). The SN0 interfered only weakly with the interaction. Lower concentrations of the two mutants were sufficient to inhibit 50% of the interaction, compared to the WT.
The D254G and K325N binding to C3(H2O) is Mg2+ and Ni2+ dependent
An ELISA assay was performed to assess whether the interaction of the recombinant FB proteins with C3 is Mg2+ and Ni2+ dependent (Fig. S4). The binding of the three proteins was significant and dose dependant in presence of Mg2+. The signal was strongly increased in presence of Ni2+. Divalent ions chelateur EDTA abrogated the binding.
Characterization of the phenotype of the endothelial cells populations
The quiescent adherent cells, the spontaneously detached cells for 24h and the cells detached in presence of 400nM staurisporine for 3h were compared fro their properties to bind Annexin V and to uptake propidium iodide. As demonstrated in Fig. S5, the quiescent cells were double negative, the spontaneously detached cells for 24h were double positive (therefore necrotic) and the staurosporine treated were 60-90% Annexin V positive and PI negative.
Complement deposition on apoptotic HUVEC
The cells, detached after staurosporine treatment were incubated with a FB-depleted serum, reconstituted with supernatants, containing recombinant FB WT or mutant-containing supernatants. In the presence of the two mutant forms of FB, C3 deposition on apoptotic cells was stronger than in presence of the WT FB (Fig. S6). The trend observed is similar to the one for the necrotic cells (Fig. 6).
REFERENCES
1. Dragon-Durey M-A, Blanc C, Marliot F, et al. The high frequency of Complement Factor H-Related CFHR1 Gene Deletion is restricted to specific subgroups of patients with atypical Hemolytic Uremic Syndrome J Med Genet. 2009;In press.
2. Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605-1612.
3. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, et al. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci U S A. 2007;104:240-245.
