Hemolytic Uremic Syndrome: a Factor H Mutation (E1172stop) Causes Defective Complement Control at the Surface of Endothelial Cells

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Hemolytic Uremic Syndrome: A Factor H Mutation (E1172Stop) Causes Defective Complement Control at the Surface of Endothelial Cells

Stefan Heinen,* Miha´ly Jo´zsi,* Andrea Hartmann,* Marina Noris,† Giuseppe Remuzzi,† Christine Skerka,* and Peter F. Zipfel*‡ *Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, and ‡Faculty of Biology, Friedrich Schiller University, Jena, Germany; and †Mario Negri Institute for Pharmacological Research Villa Camozzi, Ranica, Bergamo, Italy

Defective complement regulation results in hemolytic uremic syndrome (HUS), a disease that is characterized by microangiopathy, thrombocytopenia, and acute renal failure and that causes endothelial cell damage. For characterization of how defective complement regulation relates to the pathophysiology, the role of the complement regulator factor H and also of a mutant factor H protein was studied on the surface of human umbilical vein endothelial cells. The mutant 145-kD factor H protein was purified to homogeneity, from plasma of a patient with HUS, who is heterozygous for a factor H gene mutation G3587T, which introduces a stop codon at position 1172. Functional analyses show that the lack of the most C-terminal domain short consensus repeats 20 severely affected recognition functions (i.e., binding to heparin, C3b, C3d, and the surface of endothelial cells). Wild-type factor H as well as the mutant protein formed dimers in solution as shown by cross-linking studies and mass spectroscopy. When assayed in fluid phase, the complement regulatory activity of the mutant protein was normal and comparable to wild-type factor H. However, on the surface of endothelial cells, the mutant factor H protein showed severely reduced regulatory activities and lacked protective functions. Similarly, with the use of sheep erythrocytes, the mutant protein lacked the protective activity and caused increased hemolysis when it was added to factor H–depleted plasma. This study shows how a mutation that affects the C-terminal region of the factor H protein leads to defective complement control on cell surfaces and damage to endothelial cells in patients with HUS. These effects explain how mutant factor H causes defective complement control and in HUS—particularly under condition of inflammation and complement activation—causes endothelial cell damage. J Am Soc Nephrol 18: 506–514, 2007. doi: 10.1681/ASN.2006091069

emolytic uremic syndrome (HUS) is a disease that is tive pathway activation is shown by low APH50 values and the Ϫ characterized by microangiopathy, thrombocytope- appearance of C3 degradation products. Patients with D HUS nia, and acute renal failure (1). The disease is divided have a poor prognosis, and the disease usually progresses to H ϩ into a diarrhea-associated form (D HUS) that occurs in chil- end-stage renal failure. Ϫ dren and a non–diarrhea-associated form that occurs predom- Genetic analysis of patients with D HUS has identified mu- Ϫ ϩ inantly in adults (D HUS). The D form is caused mostly by tations of genes that code for individual complement regula- entero hemorrhagic Escherichia coli or shigella and is triggered tors, such as the soluble regulators factor H and factor I and the by a toxin (e.g., shiga toxin, shiga toxin–like products) that is membrane-bound regulator membrane cofactor protein (MCP) released by the pathogens. The disease causes renal defects. ϩ (CD46) (3–9). In addition, autoantibodies against the soluble Usually, patients with D HUS have a good prognosis; most regulator factor H were reported in three patients with HUS recover, and renal function returns to normal (2). The atypical (10). These regulatory proteins have in common that they con- form of HUS is free of entero hemorrhagic Escherichia coli in- trol the activity and the stability of the alternative pathway fections, occurs in children and adults, and can be sporadic or amplification convertase C3bBb (11). familial. Defective complement control is evidenced by low C3 Factor H is a central human regulator that controls comple- and normal C4 serum levels in serum. Deregulation of alterna- ment activation at the level of C3b (12–17). The secreted 150-kD factor H glycoprotein is composed exclusively of individually folding domains, which are termed short consensus repeats Received September 30, 2006. Accepted November 24, 2006. (SCR) or complement control protein module (18,19). Factor H Published online ahead of print. Publication date available at www.jasn.org. gene mutations have been observed in approximately 20% of Address correspondence to: Prof. Dr. Peter F. Zipfel, Department of Infection patients with HUS (20,21). So far, more than 70 patients with Biology, Leibniz Institute for Natural Products Research and Infection Biology, Hans Knoell Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany. Phone: mutations in the factor H gene have been published. The vast ϩ49-3641-656-900; Fax: ϩ49-3641-656-902; E-mail: [email protected] majority of the reported factor H gene mutations are heterozy-

gous; therefore, patients have one intact and one defective allele Surface Plasmon Resonance Binding (for review, see reference [22]). The C-terminus of factor H Protein–protein interactions were analyzed by surface plasmon res- represents a hotspot for mutations. A total of 70% of all muta- onance using a BIAcore 3000 system (BIAcore AB, Uppsala, Sweden) as tions are located in domains 19 and 20 of the factor H gene and described previously (24). represent either premature stop codons or single amino acid substitutions. The C-terminus includes the recognition region Cultivation of Endothelial Cells and Binding Experiments Human umbilical vein endothelial cells (HUVEC; American Type and has binding sites for ligands such as heparin, C3b, and C3d Culture Collection, Rockville, MD) express the membrane-bound reg- and to the surface of endothelial cells. ulators MCP (CD46), decay-accelerating factor (DAF) (CD55), and Given the association of factor H gene mutations in HUS, it is CD59 but not complement receptor 1 (CR1) (CD35) and were cultured of interest to elucidate how C-terminal recognition domain as described previously (24). Binding experiments with flow cytometry relates to the pathomechanism of HUS and endothelial cell and confocal microscopy were performed as described previously (25). damage. Here, we show for the first time, to our knowledge, that factor H mediates its protective, inhibitory activity at the Co-Factor Activity surface of endothelial cells. In addition, we show that a mutant Co-factor assays for fluid phase activity were performed as described factor H protein, which has the C-terminal domain SCR20 previously (26). For determination of co-factor activity on the surface, deleted and was purified from serum of a patient with HUS, HUVEC were cultivated in serum-free medium, washed thoroughly, and detached from the surface. Cells were incubated with purified displays defective recognition and regulatory functions at the truncated mutant factor H (factor H⌬20) or wild-type factor H (factor surface of endothelial cells and sheep erythrocytes but shows ␮ Hwt;2 g)for1hat37°C under gentle agitation. Then cells were normal regulatory activity in the fluid phase (i.e., plasma). washed three times with cold Dulbecco’s phosphate-buffered saline Therefore, modifications of the C-terminal recognition regions and incubated at 37°C in the presence of 120 ng of C3b (Merck Bio- of factor H impair the regulatory functions on the surface of sciences, Schwalbach, Germany) and 240 ng of factor I (Merck Bio- endothelial cells. sciences). At each time point, 2 ϫ 105 cells were sedimented, the supernatant was transferred to sample buffer and separated by SDS- PAGE, and C3b degradation fragments were detected by Western Materials and Methods blotting. The Patient The patient R043 is heterozygous for a mutation in the factor H gene Depletion of Factor H of Plasma nucleotide position G3587T within domain 20, introducing a stop Factor H–depleted human plasma was prepared using immune af- codon at position 1172. The patient tested negative for C3 nephritic finity column as described previously (27). Successful depletion was determined by Western blotting, and complement activity was con- factor. All experiments were conducted with the patient’s informed firmed by assay of lysis of sheep erythrocytes. consent. Hemolysis and renal function ameliorated after an intense course of plasma exchanges; however, in the subsequent 2 yr, the patient manifested three HUS recurrences. A renal biopsy that was Hemolysis Assay performed in February 1997 showed irreversible changes of chronic Hemolytic experiments were conducted in the presence of veronal nephropathy with severe thrombotic microangiopathy. Renal function buffered saline (144 mmol/L NaCl, 7 mmol/L MgCl2, and 10 mmol/L rapidly worsened, and in 1998, the patient started hemodialysis. In EGTA [pH 7.4]). Increasing amounts of purified factor Hwt or factor ϫ 7 1999, he received a cadaveric kidney transplant, but the graft failed H⌬20 were added to the depleted plasma together with 2 10 sheep within 1 mo because of disease recurrence. The factor H concentration erythrocytes. The mixture was treated for 30 min at 37°C. After centrif- in the plasma of this patient was elevated (908 ␮g/ml; normal range 350 ugation, hemolysis was determined by measurement of the absorbance to 800 ␮g/ml), and C3 plasma levels were reduced (0.51 mg/ml; at 414 nm. normal range 0.81 to 1.70 mg/ml). Cross-Linking The homobifunctional cross-linker dimethyladipimidate-dihydro- Purification of Mutant Factor H chloride (DMA) was used (Pierce Perbio Science, Bonn, Germany). ␮ ␮ Approximately 10 ml of serum from patient R043 was treated with Purified protein (1 g) or human plasma (1 l) was treated in cross- 13% polyethylene glycol (molecular weight 6000 Da) on ice. The pellet linking buffer (200 mmol/L triethanolamine [pH 9.0]) in the presence of 1 mmol/L DMA. The mixture was incubated at 25°C for 60 min in the was dissolved in 1 ml of buffer A (10 mmol/L sodium phosphate and dark. Reactions were stopped by addition of 10 ␮l of 1 mol/L Tris 100 mmol/L glycine [pH 7.3]) and applied to heparin affinity chroma- buffer (pH 9.0). tography (HiTrap Heparin column; GE Health Care, Freiburg, Ger- many). After extensive washing with buffer A, proteins were eluted with a linear salt gradient (30 to 500 mmol/L NaCl). Fractions of 400 ␮l SDS-PAGE and Western Blotting were collected, and factor H–containing fractions were combined, di- Human serum, individual chromatography fractions, cell superna- luted with deionized water, and subjected to ion exchange chromatog- tants, and cell lysates were separated by 10% SDS-PAGE under nonre- raphy (Resource Q column; GE Health Care) using buffer A. Again, ducing conditions (28). bound proteins were eluted with a linear salt gradient (range 30 to 600 mmol/L NaCl), and factor H–containing fractions were combined and Mass Spectroscopy concentrated. The factor H concentration was determined by ELISA The PepClean C18 Spin columns kit (Pierce Perbio Science) was used (23). to remove the contaminants from the PBS buffer in which the purified 508 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 506–514, 2007

proteins were stored. The samples were mixed 1:1 with saturated sinapinic acid (sinapinic acid in 0.1% [vol/vol], trifluoracetic acid, and ␮ H2O [2:1]). Part of the sample (1 to 3 l, depending on the protein concentration) was transferred onto the target (MTP384 ground steel; Bruker Daltonics, Bremen, Germany). Samples were let dry at room temperature (dried droplet procedure). The measurements were conducted with an Ultraflex TOF/TOF (Bruker Daltonics).

Results Identification of a Mutated Factor H Protein in the Plasma of Patient R043 Patient R043 is heterozygous for a nonsense factor H gene mutation at nucleotide position G3587T. This mutation introduces in SCR20 at position 1172, a premature stop codon.

Consequently, the mutant factor H⌬20 protein has most of SCR20 deleted. Plasma of this patient was separated by SDS- PAGE and subjected to Western blot analysis. Two bands of 150

and 145 kD were identified, representing factor Hwt and factor

H⌬20 (Figure 1A, top, lane 2). Both the 150- and 145-kD bands have similar intensities, indicating that the wild-type and mutant proteins are expressed in equal levels. Absence of the most Figure 1. Identification of mutant factor H (factor H⌬20)in C-terminal SCR domain in factor H⌬ was confirmed with an 20 serum of patient R043. Serum of the patient was separated by epitope-mapped mAb that binds within SCR20. This mAb D24 SDS-PAGE, transferred to a membrane, and analyzed by West- detected the 150-kD factor Hwt but not the mutant factor H⌬20 ern blotting. (A, top) Polyclonal antiserum raised against factor

protein (Figure 1A, bottom). These results show that the mutant H identified the 150-kD wild type factor H (factor Hwt) and an protein is expressed, secreted, and present in plasma in a trun- aberrant band of 145 kD, representing the mutant factor H⌬20. cated form. A polyclonal factor H antiserum that identifies multiple domains of the protein and that stains equally well with the mutant and wild-type proteins was used. (Bottom) MAb D24 Purification of Factor H⌬20 from Plasma Heparin affinity chromatography was used to separate factor that is specific for domain 20 of factor H identified the 150-kD factor Hwt but not the mutant factor H⌬20 protein, which lacks H⌬20 and factor Hwt proteins. The mutant factor H⌬20 eluted in this domain. (B) Reactivity of purified factor H⌬ and factor one peak, at a NaCl concentration of approximately 230 mM 20 Hwt. The purified mutant and wild-type proteins show reactiv- and factor Hwt in a second peak at approximately 300 mM NaCl ity with factor H antiserum, and factor Hwt but not mutant (data not shown). The individual fractions were separated by factor H reacts with mAB D24. Silver staining shows that both SDS-PAGE and analyzed by Western blotting. The factor H⌬20 mutant and wild-type proteins are purified to homogeneity protein was present in fractions 18 to 26, fraction 28 contained (bottom). (C) Domain structure of factor H indicating the 20 both proteins, and factions 30 to 36 contained the factor Hwt short consensus repeats (SCR; or CCP modules) of the intact

protein. Thus, factor H⌬20 binds with reduced affinity to hep- protein. The most C-terminal domain SCR20 is absent in the

arin. mutant factor H⌬20 protein and is shown by a dotted line. The

Fractions that contained either factor H⌬20 or factor Hwt binding epitope of mAB D24 is indicated, the N-terminal com- protein were combined, and the protein was purified to homo- plement regulatory region that is contained in domains SCR1 geneity. Purified proteins were free of contamination bands as through SCR4, and the C-terminal recognition region SCR19 and SCR20 are indicated by bars. determined by SDS-PAGE in combination with Western blotting (Figure 1B, top) or revealed silver staining (Figure 1B, bottom). The 145-kD band did not react with the mAb D24, confirming that this band represented the purified mutant pro- as indicated by the strong association and dissociation profile. tein (Figure 1B, middle, lane 2). Similar binding profiles were observed for C3d, the cleavage product of C3b, that is recognized by the C-terminal domain 20

Binding of Purified Mutant Factor H⌬20 to C3b and C3d of factor H. In contrast to factor Hwt, the mutant factor H⌬20 did Surface plasmon resonance was used to assay how the trun- not bind to C3d (Figure 2). These results show that deletion of

cation of SCR20 affects binding to the central complement the C-terminal domain in the factor H⌬20 protein severely af- component C3b. C3b was coupled covalently to the surface of fects binding to C3b and to C3d.

the sensor chip, and factor H⌬20 or factor Hwt was added in

equal amounts to the fluid phase. Factor H⌬20 bound weakly to Binding of Purified Mutant Factor H⌬20 to Endothelial Cells immobilized C3b, as indicated by the low and weak association Flow cytometry was used to assay binding of purified factor

and dissociation profile (Figure 2). Thus, truncation of the most H⌬20 or factor Hwt to HUVEC. Mutant factor H⌬20 bound

C-terminal domain strongly reduced binding to C3b. In con- weakly to HUVEC (mean fluorescence 4.1), and factor Hwt was

trast, factor Hwt bound with high affinity to immobilized C3b, stronger (mean fluorescence 77.0; data not shown). J Am Soc Nephrol 18: 506–514, 2007 Defective Complement Regulation in HUS 509

dosage-dependent binding of factor H to HUVEC was observed. In the patient’s serum, binding was weaker than with the control serum. A direct comparison of the median fluorescence confirmed the reduced binding of the patient’s plasma (Figure 3C).

Dimerization of Factor H The reduced binding profile that was observed with plasma of the heterozygous patient suggested an interaction of mutant

factor H⌬20 and factor Hwt. Therefore, dimer formation was

tested by chemical cross-linking. First purified factor Hwt was treated with the cross-linker DMA and analyzed by SDS-PAGE together with Western blotting. This approach identified two prominent bands of 150 and 300 kD, representing the monomeric form and a dimeric complex (Figure 4, lane 2). Upon treatment of human plasma, the same monomeric and dimeric complexes were identified (Figure 4, lanes 4 and 5). However, Figure 2. Reduced binding of mutant factor H⌬ to the central 20 when plasma of patient R043 was treated, the pattern was more complement component C3b and C3d. Binding of purified mutant complex. In addition to the monomeric 145- and 150-kD bands, factor H⌬20 and factor Hwt to immobilized C3b and C3d was representing mutant factor H⌬20 and factor Hwt, additional analyzed by surface plasmon resonance. Factor H⌬20 bound weakly to C3b and showed low binding to C3d. Binding of factor bands of approximately 290 and 300 kD were detected (Figure

Hwt to both C3b and C3d is indicated by the strong association 4, lanes 7 and 8). This pattern suggests dimer formation of both and dissociation profiles. Mutant factor H⌬20 or factor Hwt was the mutant factor H⌬20 and factor Hwt proteins. On the basis of injected into a flow cell or into a control flow cell. The profile that the intensity of the 145-kD mutant and the 150-kD wild-type was obtained in the control flow cell was subtracted. bands, it seems that the wild-type protein forms dimers more easily. Matrix-assisted laser desorption ionization–time of flight

analysis yielded for the purified factor Hwt and factor H⌬20 Confocal Microscopy monomer masses of 151,126.617 and 145,116.287 Da. The

In addition, surface attachment of factor H was visualized by masses of the dimeric forms of factor Hwt and factor H⌬20 were confocal microscopy. Binding of either mutant factor H⌬20 or 301,065.401 and 290,979.657 Da, respectively. factor Hwt was detected with an antiserum that resulted in green fluorescence. Factor H⌬20 bound weakly and factor Hwt strongly Co-Factor Activity of factor H⌬ and factor H to the endothelial cell surface. Nuclei were stained with DAPI 20 wt To assay how the factor H mutation relates to disease, we (blue color) and cell surfaces with Alexa 633–conjugated wheat compared the regulatory activity of the factor H⌬20 and factor germ agglutinin (red color). Upon merging of the channels, the Hwt proteins. In fluid phase, mutant factor H⌬20 and factor Hwt yellow signal reveals surface localization of factor H (Figure 3A). showed comparable co-factor activity as evidenced by the ap- Thus, three approaches, confocal microscopy, flow cytometry, and pearance and the intensity of the ␣Ј46- and ␣Ј43-kD bands of a biochemical assay, show reduced cell binding of mutant factor C3b (Figure 5A, factor Hwt versus factor H⌬20). C3b cleavage H⌬ and strong binding of factor H . This identifies SCR20 as the 20 wt products were detected already after 20 s, and their intensity major cell attachment domain of factor H. increased with time. Thus, in fluid phase, the mutant factor

H⌬20 and factor Hwt proteins have comparable regulatory ac- Assaying Cell Surface–Bound Proteins by Western Blotting tivities, and SCR20 is dispensable for this activity. Binding of factor H⌬20 or factor Hwt to endothelial cell surface was examined further with a biochemical method. After incubation with purified proteins, cells were lysed and surface- Co-Factor Activity on the Cell Surface attached proteins were assayed by Western blotting after SDS- HUS is associated with endothelial cell damage. To link defective cell binding with surface control, we analyzed co- PAGE separation. Reduced binding of the mutant factor H⌬20 factor activity of factor H directly on the cell surface. to the cell surface as compared with factor Hwt is indicated by the different intensities of the bands (Figure 3B, lanes 2 and 3). The endothelial cells that were used here express MCP Cells that were treated in medium alone showed no surface- (CD46), DAF (CD55), and CD59 but not CR1 (CD35; data not attached factor H (Figure 3B, lane 1). shown). First, the endogenous co-factor activity of the integral membrane complement regulators was assayed. Endothelial Binding of Serum-Derived Factor H of Patient R043 to cells were incubated with C3b and factor I, and C3b degrada- Endothelial Cells tion was followed upon separation of the cell lysate by SDS- Patient R043 is heterozygous for the E1172Stop mutation; there- PAGE in combination with Western blotting. Degradation ␣Ј ␣Ј ␣Ј fore, in plasma, both mutant factor H⌬20 and factor Hwt are products in the form of 68-, 46-, and 43-kD fragments of present (Figure 1A, top, lane 2). When serum of patient R043 and the C3b ␣Ј chain were detectable after 45 min, and their inten- that of a healthy control subject were used for cell binding, a sity increased with time (Figure 5B, top). Therefore, the endog- 510 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 506–514, 2007

Figure 3. Binding of mutant factor H⌬20 and factor Hwt to endothelial cells: Purified proteins. (A) Binding as assayed by confocal ␮ microscopy. HUVEC that were kept in serum-free medium for 24 h were incubated with 20 g of purified factor H⌬20 or factor Hwt for 2 h. After extensive washing, cells were treated first with a polyclonal factor H antiserum and a secondary Alexa 488–labeled anti-rabbit antibody antiserum (green), in combination with DAPI (blue: nuclear staining) and Texas red for

identification of cellular membranes. (B) Binding of mutant factor H⌬20 and factor Hwt to HUVEC as analyzed by Western blotting. Cells that were incubated for 1 h with the purified proteins were washed thoroughly and lysed. The cell lysate was separated by SDS-PAGE and transferred to a membrane, and factor H was identified by Western blotting. (C) Binding of factor H to endothelial

cells using serial dilutions of plasma that was derived from patient R043, which contains mutant factor H⌬20 and factor Hwt. Dosage-dependent binding was analyzed by flow cytometry. Mean fluorescence values that were obtained for the various serum dilutions are shown. f, plasma of the control individual; ࡗ, plasma of patient R043.

enous, integral membrane regulators MCP and DAF control C3 ␣Ј46-kD band showed that the activity of the mutant protein is activation at the surface of HUVEC. approximately one third that of the wild-type protein. To demonstrate a role of surface-attached factor H, we incu-

bated HUVEC with factor Hwt. In the presence of factor Hwt, Hemolysis of Sheep Erythrocytes degradation products were detected earlier, already after 2.5 In addition, a hemolysis assay with factor H–depleted min, and after 120 min, their intensity was prominent, thereby plasma was used to analyze how the E1172Stop mutation af- demonstrating that factor H displays co-factor activity on the fects the protective function of the protein. Sheep erythrocytes cell surface and that a combination of endogenous and attached were incubated in factor H–depleted human plasma, and in-

complement regulators controls C3b on the cell surface. On the creasing amounts of either purified factor Hwt or factor H⌬20

basis of the intensities of the cleavage products of factor were added. Factor Hwt has a protective effect, as demonstrated

H–treated versus untreated cells, factor Hwt enhances the reg- by the concentration-dependent decrease of hemolysis (Figure ulatory activity approximately 10-fold. Therefore, surface-at- 6). In contrast, addition of the same concentrations of factor

tached factor H contributes to C3b inactivation and mediates H⌬20 resulted in an increase in lysis; no protective effect was complement control (Figure 5B, middle). observed (Figure 6). Therefore, the loss of SCR20 in mutant

Having demonstrated a regulatory role of factor H on the factor H⌬20 results in a reduction of the protective activity on

surface, we assayed the mutant protein. Mutant factor H⌬20 sheep erythrocytes from complement-mediated lysis. protein showed a reduced regulatory activity; cleavage products were identified after 35 and 45 min (Figure 5B, bottom). Discussion The protective activity of the mutant protein was lower than Factor H gene mutations are associated with HUS; however, that of the wild-type protein. A densitometric analysis of the it is unclear how exactly such mutations cause endothelial cell J Am Soc Nephrol 18: 506–514, 2007 Defective Complement Regulation in HUS 511

Figure 4. Dimerization of factor Hwt and mutant factor H⌬20. Purified factor Hwt, serum-derived from a healthy person or patient R043, was treated with the indicated concentration of the cross-linker dimethyladipimidate-dihydrochloride. After incubation, the mixture was separated by SDS-PAGE, transferred to a membrane, and analyzed by Western blotting. damage. Here, we demonstrate for a mutant factor H protein When assayed in fluid phase, the complement regulatory that was derived from a patient with HUS severely reduced activity of mutant factor H was normal and comparable to regulatory activity on the cell surface. The mutant factor H the wild-type protein. We recently proposed a conforma- protein was purified from plasma of a heterozygous patient tional model for native factor H. In this model, factor H with HUS, with a G3587T exchange, that introduces a prema- forms an omega-type structure and has the C-terminal bind- ture stop codon at position 1172. The mutant 145-kD factor ing sites accessible, which then form the first and initial

H⌬20 protein shows defective binding to heparin, to C3b, to contact with surface-bound C3b and heparin (31). This model C3d, and to the surface of endothelial cells. The complement explains the reduced C3b binding that was observed in sur- regulatory activity of the mutant protein was comparable to face plasmon resonance (Figure 2) and the reduced regula- factor Hwt in fluid phase but was strongly reduced on the cell tory activity, as demonstrated by Figure 4B. Upon surface surfaces (HUVEC and sheep erythrocytes). These results dem- contact, the condensed factor H molecule unfolds and has onstrate a protective role of the plasma regulator factor H for the additional binding sites accessible. Apparently the rec- complement control on the surface of endothelial cells and ognition domain 20 of factor H, which makes the first and erythrocytes. initial contact, includes a C3b- and heparin-binding site (32). Factor H is the central fluid-phase complement regulator In fluid phase, interaction of factor H with C3b seems to be and, as shown here, also regulates complement activation on different from the interaction with surface-bound C3b and is the surface of endothelial cells and erythrocytes. The mutant explained by the greater flexibility of the two proteins in factor H⌬20 protein shows defective recognition functions solution or different conformations of C3b. In the hemolysis

(reduced binding to C3b, C3d, heparin, and endothelial cells; assay, factor Hwt but not factor H⌬20 showed a dosage- Figures 2 and 3). Defective cell binding of factor H was dependent, protective activity (Figure 6). observed both with purified mutant protein and with plasma Endothelial cells that were used in this study bind plasma of the heterozygous patient. In the patient’s plasma, which regulators to their surface and express endogenous membrane- contains a mixture of mutant factor H⌬20 and factor Hwt, bound complement regulators such as MCP, DAF, and CD59 but factor H levels were increased (908 ␮g/ml factor H), and not CR1 (data not shown). The integral membrane regulators both proteins are present at very similar levels (Figure 1, top, mediate complement control at the cell surface and inactivates lane 2). The reduced cell binding that was observed with C3b as evidenced by the appearance of the ␣Ј46 and ␣Ј43-kD patient plasma suggests an interaction of the mutant factor fragments (Figure 5B, top). Here, we demonstrate that attached

H⌬20 and factor Hwt proteins, which, however, seems to factor H contributes to the protective activity and cooperates to- affect the function of intact factor Hwt (Figure 3C). Factor H gether with the membrane-bound regulators. Under the experi- dimerization adds a new facet to the pathomechanism of mental conditions used here, the regulatory activity of surface- HUS in heterozygous patients. attached factor H was pronounced and increased degradation of With a cross-linking approach, dimer formation was demon- C3b approximately 10-fold. This finding is consistent with previ- strated for both factor Hwt and factor H⌬20. Factor H dimeriza- ous reports (31–34). However, the mutant factor H protein tion was reported earlier, and recently the C-terminus was showed reduced regulatory activity. During a local inflammatory suggested as the domain that mediates dimer formation (29,30). response, when complement is activated, endothelial cells require The 290-kD band that was observed in the patient’s plasma maximum protection. In this situation, a reduction of factor H after cross-linking shows that the mutant protein, which lacks attachment to cell surfaces and regulatory activity causes ineffi- domain 20, forms dimers. Oligomer formation under native cient complement control and leads to cell damage. The defective conditions was confirmed further by size exclusion chromatog- complement control described here for a mutant factor H protein raphy and by mass spectrometry (data not shown). that lacks the recognition module, has implications for the other 512 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 506–514, 2007

Figure 5. Co-factor activity of purified factor Hwt and mutant factor H⌬20. (A) Fluid phase. (Top) Co-factor activity of purified factor Hwt was assayed in fluid phase. C3b was incubated with factor I, and factor Hwt was added. Aliquots were removed at the indicated time points, inactivated, separated by SDS-PAGE, and transferred to a membrane, and the appearance of C3 fragments was analyzed by Western blotting. Co-factor activity is evidenced by disappearance of the ␣Ј chain and the appearance of the ␣Ј ␣Ј cleavage products of 68- and 43-kD (arrows). (Bottom) Co-factor activity of purified mutant factor H⌬20 in fluid phase. (B) Co-factor activity on the surface of endothelial cells. (Top) Endogenous co-factor activity on HUVEC in the absence of factor H. Cells were incubated with C3b and factor I at 37°C and, at the indicated time points, inactivated, separated by SDS-PAGE, and transferred to a membrane, and the appearance of C3 fragments was analyzed by Western blotting. (Middle) Co-factor activity of purified factor Hwt on endothelial HUVEC. Cells were incubated with C3b, factor I, and purified factor Hwt and analyzed in the same manner. (Bottom) Co-factor activity of purified mutant factor H⌬20 on the surface of endothelial cells. HUVEC were incubated with C3b and factor I, and mutant factor H⌬20 was added as a co-factor.

Atypical HUS is associated with endothelial cell damage and defective complement control as evidenced by low C3 plasma levels and increased levels of C3b degradation products. Apparently, the alternative pathway amplification convertase C3bBb is essential for this reaction. Consequently, a defective regulation of this enzyme, by increasing either its stability or its activity, enhances complement activation and results in tissue damage and HUS. This explains why in HUS, mutations of several complement regulators occur (e.g., factor H, MCP [CD46], factor I) and why autoantibodies to factor H have the same effect (5–10). So far, more than 75 Figure 6. Lysis of sheep erythrocytes. Sheep erythrocytes were patients with factor H gene defects have been reported, and incubated with factor H–depleted human plasma. Increasing the vast majority (75%) of the mutations are clustered in the concentrations of purified factor Hwt or factor H⌬20 were C-terminus (SCR19 and 20) and make the C-terminal recog- added. Erythrocyte lysis was measured by determination of the nition domain a hotspot for disease-associated mutations in absorbance of released hemoglobin at 414 nm. HUS. Although the initial trigger of HUS that causes endothelial cell damage still is unclear, the pathomechanism of C-terminal factor H mutations that frequently are observed in the disease—how endothelial cells are damaged as a result of HUS. It is likely that all mutations in SCR19 and 20 show a improper control of the alternative pathway convertase defective recognition function. C3bBb—becomes evident. J Am Soc Nephrol 18: 506–514, 2007 Defective Complement Regulation in HUS 513

Acknowledgments mic syndrome: Mutations in the C-terminus cause struc- The work of the authors is funded by the Deutsche Forschungsge- tural changes and defective recognition functions. JAmSoc meinschaft, the Kidneeds program (Cedar Rapids, IA), and the Foun- Nephrol 17: 170–177, 2006 dation for Children with Atypical HUS. 12. Weiler JM: Regulation of C5 convertase activity by proper- We thank patient R043 and his family for support of this study, Steffi din, factors B and H. Immunol Res 8: 305–315, 1989 Ha¨lbich for performing surface plasmon resonance assays, and Olaf 13. Weiler JM, Daha MR, Austen KF, Fearon DT: Control of the Scheibner for matrix-assisted laser desorption ionization–time of flight amplification convertase of complement by the plasma support. protein beta1H. Proc Natl Acad Sci U S A 73: 3268–3272, 1976 14. 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