Chapter 15 Purification and Functional Characterization of Factor I Sara C. Nilsson and Anna M. Blom Abstract Factor I (FI) is a soluble, 88 kDa glycoprotein present in plasma at a concentration of approximately 35 mg/L. FI inhibits all complement pathways as it degrades activated C4b and C3b when these are bound to a cofactor such as C4b-binding protein or factor H. Here, we describe a method for purification of FI from human plasma, which is based on affinity chromatography followed by anion exchange chromatography. We also describe a functional assay, in which activity of FI can be assessed. Key words Complement inhibitor, Factor I, Atypical hemolytic uremic syndrome

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Introduction Factor I (FI) is an 88 kDa serum glycoprotein that is expressed in the liver by hepatocytes [1], but also by other cells such as monocytes, fibroblasts, keratinocytes, and endothelial cells. The average serum concentration of FI is ~35 mg/L and increases during inflammation since FI is an acute phase protein. FI is a multidomain protein synthesized as a single polypeptide chain and processed to yield the heavy (50 kDa) and the light (38 kDa) chains [2], which are then covalently linked via a disulfide bridge (Fig. 1). The protein also undergoes glycosylation and each chain contains three N-linked glycans. FI can be expressed recombinantly in mammalian or insect cells but only partial proteolytic processing of the single chain FI precursor occurs under such conditions and only the processed protein has catalytic activity [3, 4]. The heavy chain consists of the FI membrane attack complex domain (FIMAC), a CD5-like domain (also known as the scavenger receptor cysteine-rich domain), low-density lipoprotein receptor 1 and 2 domains (LDLr1 and 2), and a small region of unknown homology. The light chain of FI comprises the serine protease (SP) domain [5] where the catalytic triad in the active

Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_15, © Springer Science+Business Media New York 2014

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Fig. 1 (a) Schematic overview of FI and its domains FIMAC, CD5, LDLr1/2, and SP. The sites for N-linked glycosylations are marked with arrows. (b) FI purified from plasma analyzed on SDS-PAGE. Five micrograms FI was analyzed on a 10 % SDS-PAGE gel and stained with Coomassie. In a non-reduced state intact FI migrates as an 88 kDa protein. Under reducing conditions the heavy chain of 50 kDa and light chain of 38 kDa become apparent

site is formed by His362, Asp411, and Ser507. FI contains 40 cysteines and 36 of them are involved in intra-domain disulfide bridges, and the remaining four cysteines (C15-C237, C309-C435) connect the FIMAC and LDLr1 domains and the SP domain with the heavy chain [6]. Recently a structure of FI has been solved by X-ray crystallography [7]. FI cleaves C4b and C3b at a few specific sites only in the presence of cofactors. Degradation of C4b is supported by C4b-binding protein (C4BP), membrane cofactor protein (MCP), and complement receptor 1 (CR1). C3b can be degraded to iC3b in the presence of factor H (FH), MCP and CR1 and then further to C3d only in the presence of CR1. Purified native FI, in the absence of cofactors, cleaves peptide aminomethyl coumarin substrates [8], which can be used to assess its amidolytic activity. Complete FI deficiency results mainly in recurrent infections with encapsulated microorganisms and sometimes in renal disease or an autoimmune disorder [9–11]. Heterozygous defects in FI lead to atypical hemolytic syndrome [3, 12–14]. The easiest way to purify FI is by using affinity chromatography with commercially available monoclonal antibody (MRC-OX21). FI is a quite robust protein and retains its activity after elution from an antibody.

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Materials

2.1 Starting Materials

MRC-OX21 mouse monoclonal (IgG1) directed against FI. The cell line producing MRC-OX21 was from European Collection of Cell Cultures (ECACC) No. 91060417. The antibody was expressed in two-compartment CELLine bioreactor (INTEGRA Biosciences) using fetal bovine serum with low IgG (Invitrogen). The antibody was then purified on a HiTrap Protein G column (GE Healthcare). 500 mL fresh frozen plasma.

2.2 Buffers and Reagents

Prepare all buffers using pure deionized water. The buffers applied on a column should be filtered through a 0.45 μm filter. When dialyzing plasma or purified FI use a membrane with a molecular weight cutoff of 10–14,000. 1. 0.1 M NaHCO3, pH 8.0. 2. Isopropanol. 3. 1 M HCl. 4. 1 M ethanolamine–HCl, pH 8.0. 5. 0.1 M glycine pH 2.2 + 0.5 M NaCl. 6. Tris-buffered saline (TBS): 50 mM Tris–HCl, 150 mM NaCl, pH 8.0. 7. 0.5 M EDTA, pH 7.4 (stock solution). 8. TBS + 0.5 mM EDTA, pH 7.4. 9. 10 % NaN3. 10. 0.5 M phenylmethylsulfonyl fluoride (PMSF) (stock in methanol, store at room temperature, toxic, see Note 1). 11. 1 M benzamidine (prepare fresh in water, see Note 2). 12. 3 M MgCl2, pH 6.8–7.0 (see Note 3). 13. 30 mM Tris–HCl, pH 9.0. 14. 30 mM Tris–HCl, 400 mM NaCl, pH 9.0. 15. 200 mM aminocaproic acid (EACA) (stock in water, store at −20 °C) + 2 M NaCl. 16. 50 mM Na-phosphate buffer, 150 mM NaCl, 15 mM EDTA pH 7.4. 17. 50 mM Na-phosphate buffer, 150 mM NaCl, 15 mM EDTA pH 7.4, 0.02 % NaN3.

2.3 Chromatography Resins

1. Lysine-Sepharose (GE Healthcare). 2. Affi-Gel 10 (Bio-Rad). 3. Mono Q column (GE Healthcare).

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2.4 Chromatography Equipment

2.5 Buffers and Reagents for Dot Blot and ELISA

1. XK 26 column (GE Healthcare). Depending on the availability, different types of chromatography equipment can be used, from advanced automated purification systems (e.g., ÄKTA systems from GE Healthcare) to much simpler alternatives, e.g., only a peristaltic pump. However, to facilitate the purification process we recommend the use of at least a peristaltic pump connected to an absorbance-measuring unit that can follow protein elution in real time and finally a fraction collector. 1. Washing solution: 150 mM Tris, 50 mM NaCl, 0.1 % Tween 20, pH 8.0. 2. Washing solution 10× stock: 1.5 M Tris–HCl, 0.5 M NaCl, 1 % Tween 20, pH 8.0. 3. Fish gelatin: 100 % stock (Norland HiPure liquid fish gelatin, Norlands Products). 4. Blocking solution: 3 % fish gelatin in washing solution. 5. Alkaline phosphatase (AP) buffer: 100 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2, pH 9.5. 6. 75 mg/mL Nitro blue tetrazolium (NBT) (stock in dimethylformamide, Fermentas). 7. 50 mg/mL 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt (BCIP-T) (stock in 70 % dimethylformamide, Fermentas). 8. MRCOX-21 antibody (monoclonal antibody against human FI). 9. Goat anti-human FI sera (polyclonal antibody against human FI, Quidel). 10. Rabbit anti-mouse Ig-AP. 11. Rabbit anti-goat Ig-horseradish peroxidase (HRP). 12. Goat anti-mouse Ig-HRP. 13. 1,2-phenylenediamine dihydrochloride (OPD) tablets (Dako). 14. 0.5 M H2SO4. 15. TBS: 50 mM Tris–HCl, 150 mM NaCl, pH 8.0. 16. HRP buffer for western blot: 10 mg diaminobenzidine (DAB), 100 mL TBS, 1 mL 0.06 M NiCl2, 10 μL H2O2.

2.6 Buffers and Materials for Assay Testing Function of FI

1. Veronal buffer (VBS) stock (25 mM): 9.0 mM Na-barbituric acid, 15.5 mM barbituric acid, and 720 mM NaCl pH 7.35 (see Note 4). 2. GVB–Ni: 0.1 % gelatin, 2.5 mM VBS, 100 mM NaCl, and 2.5 mM NiCl2 (see Note 5). 3. GVB–EDTA: 0.1 % gelatin, 2.5 mM VBS, 100 mM NaCl, and 2 mM EDTA (see Note 5).

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4. DGVB++: 0.1 % gelatin, 2.5 mM VBS, 140 mM glucose, 1 mM CaCl2, and 0.15 mM CaCl2 (see Note 5). 5. Flow cytometry medium (FCM): PBS, 1 % bovine serum albumin, 15 mM EDTA, and 30 mM NaN3. 6. TBS: 50 mM Tris–HCl, 150 mM NaCl, pH 8.0. 7. Sheep erythrocytes (Complement Technology Ltd). 8. Purified C3, factor B (FB), and factor D (FD) (Complement Technology). 9. FI and FH can be purified from plasma or bought from Complement Technology. 10. V-bottom plate with 96 wells (Nunc). 11. Mouse anti-human C3d (Quidel). 12. Mouse anti-human iC3b (Quidel). 13. Goat anti-mouse Ig–fluorescein isothiocyanate (FITC) (Goat F(ab’)2 (Dako). 2.7 Equipment Required for Assay Testing Function of FI

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1. Thermomixer (e.g., Eppendorf). 2. Flow cytometer.

Methods

3.1 Coupling of MRC-OX21 to Affi-Gel 10

Affi-Gel 10 (Bio-Rad) is a N-hydroxysuccinimide ester of a derivatized cross-linked agarose gel bead support and couples to ligands spontaneously in aqueous solution. Ether bonds link the spacer arm to chemically cross-linked agarose gel beads while amide bonds couple the protein ligand to the terminal carboxyl of the spacer arm. The gel is resistant to urea, guanidine HCl, heat, solvents, acidic and basic conditions (pH 2–11). 1. Dialyze MRC-OX21 (see Note 6 for amount) against 0.1 M NaHCO3, pH 8.0. Dialyze first against 2 L buffer for 2 h and then overnight with additional 3 L. After dialysis, measure the absorbance at 280 nm. 2. Gently shake the Affi-Gel 10 vial and transfer ~50 mL to a Buchner funnel. 3. Wash rapidly, using vacuum, with 3 volumes isopropanol and then with 3 volumes of cold deionized H2O. 4. Transfer the gel to a beaker and add the dialyzed MRC-OX21 and incubate with gentle agitation for 1.5–2 h at room temperature (see Note 7). 5. To block the remaining esters add 0.1 mL/1 mL gel of freshly made 1 M ethanolamine–HCl, pH 8.0. Incubate with gentle agitation for 1 h at room temperature.

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6. Transfer the gel to a XK-26 column and continue working at 4 °C. 7. Wash the column with 5 column volumes (CV) of 0.1 M NaHCO3, pH 8.0 using a flow of 2 mL/min. 8. Continue washing with 5 CV of 0.1 M glycine pH 2.2 supplemented with 0.5 M NaCl. 9. Equilibrate the column with 5 CV of TBS + 0.5 mM EDTA pH 7.4. 10. If the column is not to be used immediately, store it at 4 °C (see Note 8). 3.2 Purification of FI on the MRC-OX21 Column

The purification of FI should be performed at 4 °C. 1. The MRC-OX21 column is prepared as described above (see Subheading 3.1). 2. Add protease inhibitors, 5 mM EDTA, 1 mM PMSF, and 10 mM benzamidine, to 500 mL human plasma (preferably fresh frozen) (see Notes 1 and 2). 3. Dialyze the plasma against TBS + 0.5 mM EDTA pH 7.4 (2 × 10 L) at 4 °C, overnight (see Note 7). 4. Filter the dialyzed plasma through a 0.45 μm filter. If there is a visible precipitate it is advisable to centrifuge the plasma first for 20 min at 5,000 × g (see Note 9). 5. Equilibrate the MRC-OX21 column with 5 CV of TBS + 0.5 mM EDTA pH 7.4. 6. Load 100 mL filtered plasma on the column with a flow of 2 mL/min. Collect the flow through and check with a dot blot (see Subheading 3.3.1) if most of the FI is bound to the column. If not the plasma can be loaded once more on the column. 7. Wash the column with 6 CV of TBS + 0.5 mM EDTA pH 7.4 to remove unbound proteins. 8. Elute FI from the MRC-OX21 column with 5 CV of 3 M MgCl2 pH 6.8–7.0. Collect fractions of 8 mL. 9. Pool the fractions that contain FI. The fractions can be run on a SDS-PAGE and/or western blot to analyze the yield and purity of the FI (see Subheadings 3.3.3 and 3.3.4). FI migrates as 88 kDa (Fig. 1b) or 50 kDa and 38 kDa (Fig. 1b) band under non-reducing and reducing conditions, respectively. If the FI is pure, dialyze against TBS at 4 °C, overnight. Freeze in aliquots at −80 °C. Repeat the purification four more times to us up the whole prepared plasma. 10. If FI is not pure enough it can be further purified on a Mono Q column, which is a strong anion exchanger. 11. Dialyze the FI-containing fractions against 30 mM Tris–HCl pH 9.0.

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12. Equilibrate the Mono Q column with 5 CV of 30 mM Tris– HCl pH 9.0, with a flow of 1 mL/min. 13. Apply the dialyzed FI to the Mono Q column and wash with 10 CV 30 mM Tris–HCl pH 9.0. 14. Elute FI with a 50 mL gradient to 400 mM NaCl in 30 mM Tris–HCl pH 9.0. Collect fractions of 1 mL. FI elutes in the middle of the gradient. Separate the fractions on a SDS-PAGE and stain with Coomassie Brilliant Blue or silver salts. 15. Pool the fractions containing FI and dialyze against the desired buffer or freeze directly in aliquots at −80 °C. Avoid repeated freezing and thawing. 3.3

Detection of FI

3.3.1 Dot Blot

An easy and fast method to analyze if a sample contains FI is a dot blot. 1. Load 3–5 μL of each sample on an ultra bind US450 membrane (Pall Life Sciences). 2. Block nonspecific binding sites on the membrane by incubating with blocking solution for 30 min. 3. Add 1 µg/mL MRC-OX21 in blocking solution and incubate for 30–60 min at room temperature with gentle agitation. 4. Wash the membrane with washing solution for 3 × 5 min. 5. Add a secondary antibody, e.g., polyclonal rabbit anti-mouse conjugated to alkaline phosphatase (Dako), diluted 2,000× in blocking solution and incubate as before (see Note 10). 6. Wash the membrane with washing solution for 3 × 5 min. 7. Develop the blot by adding AP buffer supplemented with 330 μg/mL NBT (Fermentas) and 165 μg/mL BCIP-T (Fermentas) (see Note 11). Stop the development by rinsing the membrane with water.

3.3.2

ELISA

The concentration of FI can be measured using ELISA using purified FI or normal human serum/plasma as a standard. The volume is 50 μL per well in all steps if not stated otherwise. 1. Coat MaxiSorp plates (Nunc) with polyclonal goat anti-human FI sera (Quidel) diluted 1,000× in 50 mM sodium carbonate buffer pH 9.5, at 4 °C overnight. 2. Wash the plate four times with the washing solution (300 μL/ well). 3. Block the wells with 250 μL blocking solution for 1 h at 37 °C. 4. Add the dilutions of FI of serum/plasma (a standard curve) prepared in blocking solution and only blocking solution as a negative control, incubate for 1 h at 37 °C. 5. Wash the plate four times with the washing solution (300 μL/ well).

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6. Add 1 μg/mL MRC-OX21 diluted in blocking solution and incubate for 1 h at 37 °C. 7. Wash the plate four times with the washing solution (300 μL/ well). 8. Add polyclonal goat anti-mouse antibody conjugated to HRP diluted 2,000× in blocking solution and incubate for 1 h at 37 °C. 9. Wash the plate four times with the washing solution (300 μL/ well). 10. Develop the plate by adding 2 OPD tablets dissolved in 6 mL water and 2.5 μL H2O2. 11. Stop the reaction with 0.5 M H2SO4 and measure the absorbance at 490 nm. 3.3.3 SDS-PAGE

FI can be analyzed on a 10 % SDS-PAGE gel and stained with Coomassie Brilliant Blue (Fig. 1b) or silver salts. Under nonreducing conditions FI migrates as a single band of 88 kDa and at reducing conditions it migrates as a heavy chain of 50 kDa and light chain of 38 kDa.

3.3.4 Western Blot

FI can also be analyzed using western blotting. For non-reducing conditions MRCOX-21 followed by goat anti-mouse antibodies are preferable to use and for reducing conditions goat anti-human FI followed by polyclonal rabbit anti-goat antibodies are a good choice (see Note 12).

3.4 Functional Assay for FI

The activity of FI can be determined using fluid-phase C3b and C4b degradation assay (see Chap. 14) or membrane bound C3b as a substrate in presence of a cofactor (e.g., C4BP, FH, MCP, or CR1) [15]. In the assay using membrane bound C3b, sheep erythrocytes are coated with C3b, which is then cleaved by FI together with FH. Two monoclonal antibodies, anti-C3d and anti-iC3b, are used to detect the cleavage products of C3b using flow cytometry (Fig. 2). Most anti-C3 antibodies recognize to various extent many forms of C3 such as C3b, iC3b and C3d and therefore it is advisable to use a ratio of the geometric means for iC3b–C3d to estimate activity of FI. Anti-C3d antibody determines how much C3b was deposited in total on the surface of the erythrocytes and antiiC3b antibody how much iC3b is formed after cleavage with FI and FH. A high ratio of iC3b–C3d indicates degradation of FI. 1. Wash ~3 mL sheep erythrocytes (1 × 109 cells/mL) 3–5 times in 10 mL ice-cold GVB–Ni until the supernatant is transparent. Between each wash centrifuge the erythrocytes for 800 × g for 5 min. After the last washing resuspend the pellet (~0.5 mL) in 5.5 mL GVB–Ni.

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a

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Fig. 2 C3b was deposited on the surface of sheep erythrocytes by incubation with C3, FB, and FD. (a) A histogram shows the amount of iC3b and C3d present on the erythrocytes after incubation with 0.1, 0.5, and 1.0 μg/mL FI together with 20 μg/mL FH. (b) The amount of deposited C3b and liberated iC3b was detected using specific antibodies and the results are presented as a ratio iC3b-C3d

2. Transfer 200 μL erythrocytes (from the 6 mL solution) to an eppendorf tube and add to a final concentration of 0.2 mg/ mL C3, 0.02 mg/mL FB, and 0.4 μg/mL FD and incubate in a thermomixer at 800 rpm for 30 min at 30 °C. 3. Spin down the erythrocytes in a tabletop centrifuge for 3 min at 800 × g. Wash the erythrocytes twice with 1 mL ice-cold GVB–EDTA. Resuspend the pellet with 200 μL ice-cold GVB-EDTA. 4. Incubate the erythrocytes (200 μL) with 0.02 mg/mL FB and 0.4 μg/mL FD for 5 min at 30 °C, 800 rpm. 5. Wash the erythrocytes GVB–EDTA.

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6. Incubate the erythrocytes (200 μL) with 0.2 mg/mL C3 for 20 min at 30 °C, 800 rpm.

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7. Wash the cells erythrocytes with 1 mL ice-cold GVB–Ni. 8. Incubate the erythrocytes (200 μL) with 0.02 mg/mL FB and 0.4 μg/mL FD for 5 min at 30 °C, 800 rpm. 9. Wash the erythrocytes twice with 1 mL ice-cold GVB–EDTA. 10. Incubate the erythrocytes (200 μL) with 0.2 mg/mL C3 for 20 min at 30 °C, 800 rpm. 11. Wash the erythrocytes twice with 1 mL ice-cold DGVB++ and resuspend the pellet in 5 mL DGVB++. Transfer 100 μL erythrocytes to each well in a 96-well V-bottom plate. Spin down the cells at 800 × g for 3 min and discard the supernatant. 12. Resuspend and incubate the erythrocytes with 25 μL FI and 25 μL FH for 60 min at 37 °C, 800 rpm (see Note 13). 13. Wash the erythrocytes 3 times in 200 μL FCM and split the erythrocytes to two wells. Transfer 100 μL to each well and spin down the cells. 14. Incubate the erythrocytes with monoclonal antibodies against C3 for 30 min at 4 °C. In one well incubate with 50 μL mouse anti-human C3d diluted 200× in FCM and in the other well with 50 μL mouse anti-human iC3b diluted 200× in FCM. 15. Wash the erythrocytes 2–3 times with FCM. 16. Incubate the erythrocytes for 30 min at 4 °C with 50 μL goat anti-mouse Ig-FITC diluted 100× in FCM. 17. Wash the erythrocytes twice in FCM (see Note 14). 18. Analyze the erythrocytes on a flow cytometer (see Note 15).

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Notes 1. PMSF is toxic and can be exchanged for 4-(2-aminoethyl)benzene-sulfonyl fluoride (AEBSF, Calbiochem), which is more expensive but less toxic and more stable alternative. Since PMSF is rapidly degraded in water, the stock solution should be in methanol. 2. Benzamidine is inactivated by oxidation and should therefore be prepared fresh before use. 3. To adjust the pH add a Tris base. 4. First dissolve barbituric acid in hot H2O (~100 mL at 150– 200 °C), let it cool down, and then add to another beaker with the rest of the components dissolved. Sterile filter and store at 4 °C. Use the buffer when it is ice-cold. 5. When preparing buffer containing gelatin first boil the gelatin in 10–15 mL H2O. When it is dissolved let it cool down and then add the rest of the chemicals to the solution.

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6. It is advisable to couple 2–10 mg protein per mL gel. A column of 50 mL is a preferable size of choice, requiring 250 mg of antibody for coupling. 7. The sample added should be at least 0.5 mL ligand solution per milliliter of gel. Measure the absorbance at 280 nm of the supernatant to estimate how much of the protein that has bound to the gel. Before measuring the absorbance, the pH of the sample should be lowered by adding 1 M HCl to a final concentration of 0.01 M HCl because at neutral or basic pH the N-hydroxysuccinimide released during the coupling will absorb at 280 nm. 8. Addition of 0.02 % NaN3 will prevent bacterial growth in the column. 9. It is optional to remove plasminogen from plasma before applying it on the MRC-OX21 column. Plasminogen is a zymogen of the active serine protease plasmin and can be activated by several different enzymes such as tissue/urokinase plasminogen activator, kallikrein, and factor XII. Plasmin may eventually degrade many plasma proteins during the purification including FI. Therefore the following step may be added in case one experiences problems with degradation. (a) Wash 100 mL lysine-Sepharose on a sintered glass funnel with 200 mL of 200 mM EACA + 2 M NaCl and then with 500 mL of 50 mM Na-phosphate buffer, 150 mM NaCl, 15 mM EDTA, pH 7.4. (b) Add 100 mL of lysine-Sepharose to the plasma (plasminogen will bind to the gel) and incubate for 1 h at 4 °C with gentle agitation (do not use magnetic stirrer). Centrifuge for 20 min at 2,830 × g, 4 °C to remove the gel. The supernatant contains FI, which can be further applied on a MRC-OX21 column (see Subheading 3.2). (c) Regenerate the gel with 200 mL of 200 mM EACA + 2 M NaCl, plasminogen will be eluted. Wash the gel with 500 mL of 50 mM Na-phosphate buffer, 150 mM NaCl, 15 mM EDTA, pH 7.4, and 0.02 % NaN3 (see Note 8). 10. A polyclonal antibody against FI (e.g., goat anti-human FI, Quidel) can also be used to detect FI on a dot blot. 11. A secondary antibody conjugated to HRP also works for detection on dot blot, but then a different development buffer is needed. Mix 10 mg DAB with 100 mL TBS, when it is dissolved add 1 mL 0.06 M NiCl2 and 10 μL H2O2. Add the solution to the membrane. Stop the development by rinsing the membrane with water. 12. The light chain of FI is usually detected with lower intensity compared to the heavy chain when using polyclonal antibodies.

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13. Dilute FI and FH in TBS and make two times the concentration due to the dilution factor. The final volume should be 50 μL. Optimization of the concentrations is required but it is advisable to test concentrations in the range of 0.5–10 μg/mL FI and 5–20 μg/mL FH. 14. The final volume of the samples depends on the type of flow cytometer, which is used to analyze the samples. When analyzing many samples, it is preferable to use a 96-well plate autosampler and then a volume of 150 μL per well may be sufficient. 15. To be able to adjust the settings of the flow cytometer, analyze untreated erythrocytes before you run the actual samples. References 1. Morris KM, Aden DP, Knowles BB et al (1982) Complement biosynthesis by the human hepatoma-derived cell line HepG2. J Clin Invest 70:906–913 2. Goldberger G, Arnaout MA, Aden D et al (1984) Biosynthesis and postsynthetic processing of human C3b/C4b inactivator (factor I) in three hepatoma cell lines. J Biol Chem 259:6492–6497 3. Nilsson SC, Karpman D, Vaziri-Sani F et al (2007) A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation. Mol Immunol 44:1835–1844 4. Ullman CG, Chamberlain D, Ansari A et al (1998) Human complement factor I: its expression by insect cells and its biochemical and structural characterisation. Mol Immunol 35:503–512 5. Chamberlain D, Ullman CG, Perkins SJ (1998) Possible arrangement of the five domains in human complement factor I as determined by a combination of X-ray and neutron scattering and homology modeling. Biochemistry 37:13918–13929 6. Tsiftsoglou SA, Willis AC, Li P et al (2005) The catalytically active serine protease domain of human complement factor I. Biochemistry 44:6239–6249 7. Roversi P, Johnson S, Caesar JJ et al (2011) Structural basis for complement factor I control and its disease-associated sequence polymorphisms. Proc Natl Acad Sci USA 108:12839–12844 8. Tsiftsoglou SA, Sim RB (2004) Human complement factor I does not require cofactors for

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cleavage of synthetic substrates. J Immunol 173:367–375 Amadei N, Baracho GV, Nudelman V et al (2001) Inherited complete factor I deficiency associated with systemic lupus erythematosus, higher susceptibility to infection and low levels of factor H. Scand J Immunol 53:615–621 Nilsson SC, Trouw LA, Renault N et al (2009) Genetic, molecular and functional analyses of complement factor I deficiency. Eur J Immunol 39:310–323 Sadallah S, Gudat F, Laissue JA et al (1999) Glomerulonephritis in a patient with complement factor I deficiency. Am J Kidney Dis 33:1153–1157 Bienaime F, Dragon-Durey MA, Regnier CH et al (2010) Mutations in components of complement influence the outcome of Factor I-associated atypical hemolytic uremic syndrome. Kidney Int 77:339–349 Esparza-Gordillo J, Jorge EG, Garrido CA et al (2006) Insights into hemolytic uremic syndrome: segregation of three independent predisposition factors in a large, multiple affected pedigree. Mol Immunol 43:1769–1775 Kavanagh D, Kemp EJ, Mayland E et al (2005) Mutations in complement factor I predispose to development of atypical hemolytic uremic syndrome. J Am Soc Nephrol 16:2150–2155 Nilsson SC, Kalchishkova N, Trouw LA et al (2010) Mutations in complement factor I as found in atypical hemolytic uremic syndrome lead to either altered secretion or altered function of factor I. Eur J Immunol 40:172–185