Factor H and Atypical Hemolytic Uremic Syndrome: Mutations in the C-Terminus Cause Structural Changes and Defective Recognition Functions
Mihály Józsi*,
Stefan Heinen*,
Andrea Hartmann*,
Clemens W. Ostrowicz*,
Steffi Hälbich*,
Heiko Richter*,
Anja Kunert*,
Christoph Licht,
Rebecca E. Saunders,
Stephen J. Perkins,
Peter F. Zipfel*, and
Christine Skerka*
* Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany; Children’s Hospital of the University of Cologne, Pediatric Nephrology, Cologne, Germany; Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, University College London, London, United Kingdom; and Frierich Schiller University, Jena, Germany
Address correspondence to: Dr. Christine Skerka, Department of Infection Biology, Leibniz Institute for Natural Product Research, and Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany. Phone: +49-3641-656848; Fax: +49-3641-656902; E-mail: christine.skerka{at}hki-jena.de
Received for publication August 19, 2005.
Accepted for publication October 15, 2005.
Atypical hemolytic uremic syndrome is a disease that is characterizedby microangiopathic hemolytic anemia, thrombocytopenia, andacute renal failure. Mutations in the complement regulator factorH are associated with the inherited form of the disease, and>60% of the mutations are located within the C terminus offactor H. The C-terminus of factor H, represented by short consensusrepeat 19 (SCR19) and SCR20, harbors multiple functions; consequently,this study aimed to examine the functional effects of clinicallyreported mutations in these SCR. Mutant factor H proteins (W1157R,W1183L, V1197A, R1210C, R1215G, and P1226S) were recombinantlyexpressed and functionally characterized. All six mutant proteinsshowed severely reduced heparin, C3b, C3d, and endothelial cellbinding. By peptide spot analyses, four linear regions thatare involved in heparin, C3b, and C3d binding were localizedin SCR19 and SCR20. A three-dimensional homology model of thetwo domains suggests that these four regions form a common bindingsite across both domains. In addition, this structural modelidentifies two types of residues: Type A residues are positionedon the SCR surface and are represented by mutants W1157R, W1183L,R1210C, and R1215G; and type B residues are buried within theSCR structure and affect mutations V1197A and P1226S. Mutationsof both types of residue result in the same functional defects,namely the reduced binding of factor H to surface-attached C3bmolecules and reduced complement regulatory activity at thecell surfaces. The buried type B mutations seem to affect ligandinteraction of factor H more severely than the surface-exposedmutations.
Hemolytic uremic syndrome (HUS) is a disease that is characterizedby microangiopathic hemolytic anemia, thrombocytopenia, andacute renal failure (reviewed in [1]). The typical epidemic-or diarrhea-associated form of HUS, which is nonrecurring andshows almost complete recovery, is most common in infants andyoung children. A second form, termed atypical, non–diarrhea-associatedHUS (aHUS), occurs predominantly in adults. This form is rare,is often recurrent, and generally has a poor outcome. Most patientswith aHUS develop end-stage renal failure and require long-termdialysis. Several studies have shown that aHUS is associatedwith defective complement regulation, which is caused by geneticalterations of complement regulators or by autoantibodies toindividual regulators (2). The majority of aHUS mutations (65cases) have been identified in the multidomain complement regulatorfactor H (3–8). Five patients showed mutations withinthe membrane co-factor protein (CD46) (9,10), four patientshad mutations in the serine protease factor I (11,12), and forthree aHUS patients, autoantibodies for factor H have been reported(13).
Factor H is a central complement regulator that is composedof 20 short consensus repeat (SCR) domains, in which the complementregulatory region is located in the N-terminal SCR1 to SCR4,whereas its cell binding recognition domain is located in itsC-terminal part (14). Two separate binding sites for heparinand sialic acid have been identified in SCR7 and SCR19/SCR20,whereas a third site may exist in SCR13 (15–19). The SCR19and SCR20 form the major recognition region of the protein (20,21).In support of this conclusion, functional analyses of recombinantfactor H proteins mutated in SCR19/SCR20 at sites identifiedin patients with HUS display limited capacities to bind to cellsurfaces (21). Moreover, a mAb with an epitope spanning SCR19and SCR20 inhibits heparin binding by factor H (14,22). Morethan half of the HUS-associated mutations are clustered in SCR20and SCR19, and different residues are affected (1–5).So far, defective C3b and endothelial cell binding has beendemonstrated for two mutant proteins (4,21). It therefore isof interest to analyze whether additional C-terminal mutantsresult in the same or at least similar defects.
To correlate the reported aHUS mutations with factor H recognitionfunctions, we used peptide spot assays to localize the linearbinding regions in SCR19 and SCR20. In addition, recombinantfactor H proteins that incorporate six distinct aHUS-associatedmutations were expressed and used for functional analyses. Molecularmodeling studies of SCR19 and SCR20 were performed to providestructural insights of the mutations. This approach identifiedin SCR19 and SCR20 a total of four linear, mostly overlappingregions that are responsible for binding to the various ligands.All six mutant proteins showed defective ligand and cell binding.These results demonstrate that defective factor H is relateddirectly to aHUS.
Peptide Spot Analysis
Forty peptides that represent SCR19 and SCR20 of factor H (residues1104 to 1231) with lengths of 13 amino acids and an overlapof 10 amino acids were synthesized and coupled to a cellulosemembrane (Jerini Peptide Technologies, Berlin, Germany). Themembranes were treated and analyzed as described (23).
Generation of Mutant Factor H Fragments
Single nucleotide exchanges in SCR20, representing the W1157R,W1183L, V1197A, and P1226S substitutions, were introduced bythe QuikChange site-directed mutagenesis technique (Stratagene,La Jolla, CA) according to the manufacturer’s instructions.The following primers were used to generate mutant Factor Hsequences in the expression vector pBSV-FH8–20 (24) (modifiednucleotides in the sequence are in boldface):
ForW1157R 5'-C A T G T A G A A A T G G A C A A C G G T C A GA A C C A C C A A A A T G C-3'
RevW1157R 5'-G C A T T T T G G T G G T T C T G A C C G T T GT C C A T T T C T A C A T G-3'
ForW1183L 5'-C A T A G C A T T A A G G T T G A C A G C C A AA C A G A A G C T T T A T T C G-3'
RevW1183L 5'-C T G T T T G G C T G T C A A C C T T A A T G CT A T G T T A T A A T T T T C C-3'
ForV1197A 5'-C G A G A A C A G G T G A A T C A G C T G A A TT T G T G T G T A A A C G G-3'
RevV1197A 5'-C C G T T T A C A C A C A A A T T C A G C T G AT T C A C C T G T T C T C G-3'
ForP1226S 5'-C A T G T T G G G A T G G G A A A C T G G A G TA T T C A A C T T G T G C A A A A A G-3'
RevP1226S 5'-C T T T T T G C A C A A G T T G A A T A C T C CA G T T T C C C A T C C C A A C A T G-3'
The sequence of the various mutant vectors was verified by sequenceanalysis. The generation of the wild-type factor H fragmentSCR8 to SCR20 and mutants R1210C (FH8-20/R1210C) and R1215G(FH8-20/R1215G) was described previously (21).
Insect Cell Culture Spodoptera frugiperda Sf9 cells were cultivated as described(24). Cells were infected with recombinant virus, and purificationof the recombinant proteins was performed according to standardprocedures (24)
Heparin Chromatography of Mutant Proteins
The binding of the recombinant mutant and wild-type factor Hproteins to a heparin matrix was analyzed using an ÄKTAPrimesystem (GE Health Care, Uppsala, Sweden). Supernatant that wasprepared from infected insect cells was diluted in 0.5 x PBS,and 5 to 10 ml of this solution was applied to a heparin matrix(HiTrap; GE Health Care) at a flow rate of 1 ml/min. After loading,the matrix was washed with 100 ml of 20 mM NaCl in 0.5 x PBS.Subsequently, bound proteins were eluted using a linear saltgradient that ranged from 100 to 500 mM NaCl. Fractions of 500µl were collected, separated by SDS-PAGE, and used forimmunoblotting. The elution profiles of the various proteinswere compared using the Prime View software (GE Health Care).For each protein, this separation was repeated three times,and in all cases, identical elution profiles were obtained.
Binding to C3b and C3d by Surface Plasmon Resonance
The binding to C3b and C3d of mutant recombinant and wild-typefactor H SCR8-SCR20 was assayed using a Biacore 3000 instrument(21). Briefly, C3b or C3d was immobilized via standard aminecoupling to the flow cells of a sensor chip (carboxylated dextranchip CM5; Biacore, Uppsala, Sweden). The surface of the flowcells was activated, and C3b or C3d was diluted in couplingbuffer (10 mM acetate buffer [pH 5.0]) was injected until anappropriate level of coupling was reached (approximately 4000resonance units). Each analyte was injected separately intoa flow cell coupled with C3b or C3d or into a blank controlusing a flow rate of 5 µl/min at 25°C. Each bindingexperiment was performed three times.
ELISA to Determine C3b and C3d Binding
MaxiSorp plastic plates (Nunc, Wiesbaden, Germany) were coatedwith 10 µg/ml C3b or C3d (Calbiochem-Novabiochem Corp.,San Diego, CA) in PBS. After blocking with 3% BSA in PBS, thevarious factor H fragments were added. Binding of the factorH proteins was detected by serial incubation with goat anti-humanfactor H (Calbiochem-Novabiochem Corp.) diluted 1:1000 in PBSthat contained 0.05% Tween-20 and horseradish peroxidase–conjugatedrabbit anti-goat Ig 1:1000 (Dako, Hamburg, Germany). The reactionwas developed using 100 µg/ml 3'3'5'5'-tetramethylbenzidine(Roth, Karlsruhe, Germany) in 0.1 M Na-acetate buffer (pH 5.5)as chromogene, 2 µl/ml 30% H2O2 was used as substrate,and the color reaction was stopped with 2 M H2SO4. The absorbancewas measured at 450 nm on a Spectra Max 190 photometer (MolecularDevices, Eugene, OR).
Cultivation of Endothelial Cells and Binding Assay
Human umbilical vein endothelial cells (American Type CultureCollection, Rockville, MD) were grown as described (21). Forbinding experiments, cells were grown to confluence either incell culture flasks or on Lab-Tek chamber slides (Nalgene-NuncInternational, Wiesbaden, Germany), washed, and cultivated inserum-free DMEM for 20 to 30 h.
SDS-PAGE and Western Blot Analysis
Purified recombinant proteins were separated by SDS-PAGE undernonreducing conditions as described (25).
Immunofluorescence Staining
Endothelial cells that were grown on eight-well chamber slidesfor 24 h in FCS-free medium were washed and fixed with 4% paraformaldehydefor 30 min. Cells were treated with 1% BSA/PBS for 30 min toprevent unspecific binding and incubated for 1 h with purifiedrecombinant protein diluted in 0.5 x PBS using identical amountsof recombinant wild-type factor H SCR8-SCR20 or mutant protein(W1157R, W1183L, V1197A, R1210C, and R1215G). Cells were incubatedovernight with polyclonal anti–factor H antiserum (Calbiochem-Novabiochem)diluted 1:200 in blocking buffer. Excess antibody was removedby washing with PBS, and the cells were incubated further atroom temperature with an Alexa fluor-488–conjugated donkeyanti-goat antibody (Molecular Probes, Eugene, OR). After 2 h,cells were washed with PBS and mounted in fluorescence-preservingmedium. The nuclei were stained with DAPI (10 µg/ml; SigmaAldrich, Taufkirchen, Germany) and the cell wall with Alexa633–labeled wheat germ agglutinin (Molecular Probes).Fluorescence staining was visualized with appropriate filtersettings using a confocal laser scanning microscope (Zeiss-LSM-510META; Carl Zeiss, Jena, Germany) equipped with the META detector.
Homology Modeling
The homology modeling of the factor H SCR domains using theMODELLER program and the PROCHECK verification program is describedelsewhere (26). The models for SCR19 and SCR20 were based onthe crystal structures of SCR3 and SCR1, respectively, in vacciniavirus coat control protein (VCP) (27) because these showed thehighest sequence identity (40 and 18%, respectively, with SCR19and SCR20) and the minimum number of insertions and deletions.The SCR19 and SCR20 models are available from the factor H aHUSweb database (http://www.FH-HUS.org) (26). Whereas the secondarystructure is expected to be conserved and modeled accurately,individual sidechains may not be positioned correctly in allcases. SCR19 and SCR20 are joined by a short three-residue linker,but this linker was not modeled because of the great variabilityin conformation seen between adjacent SCR domains (28).
Peptide Spot Analysis
Peptide spot analyses were performed to identify within theC-terminus of factor H the linear binding regions and aminoacids that are relevant for interactions with the various ligands.Peptides that represent the sequence of SCR19 and SCR20 werespotted and probed with heparin, C3b, and C3d. These analysesidentified four linear regions that interact with heparin (Figure 1A,top). Region I is 10 residues long (amino acids 1145 to1154), is located in SCR19, and binds relatively weakly. Threemajor binding sites were located in SCR20 (region II, aminoacids 1178 to 1187; region III, amino acids 1202 to 1215; andregion IV, residues 1229 to 1231; Figure 1B). Incubation ofa membrane with an unrelated control protein showed no positivespots (data not shown). In addition, separate membranes wereused to map the interaction sites for C3b and C3d (Figure 1A,middle and bottom). This approach identified the same or verysimilar regions for the three ligands and revealed a substantialoverlap of the binding sites, together with evidence of ligandspecificity. C3b binding is mediated by regions II, III, andIV, whereas C3d binding is meditated by regions I, II, and III.It also seems that for heparin binding, a longer stretch ofregion II is used as for C3b and C3d binding. Hence, regionIV binds heparin and C3b but not C3d. Thus, SCR19 and SCR20of factor H use similar and overlapping residues for bindingto the three ligands.
Figure 1. Localization of linear binding motifs in the C-terminal recognition region of factor H. (A) Peptides with a length of 13 amino acids and an overlap of 10 amino acids that covered the complete short consensus repeat 19 (SCR19) and SCR20 domains of factor H were synthesized; spotted onto the membrane; and analyzed for the binding of heparin, C3b, and C3d. The four identified binding regions are boxed. (B) Linear protein sequences of SCR19 and SCR20 of factor H. The four binding regions identified by peptide spot analyses are boxed, and the atypical hemolytic uremic syndrome (aHUS)-associated mutations with their corresponding amino acid exchanges are shown. The circled amino acids were those studied in this work. Structural cysteins are marked by gray shading. The double mutations at S1181 and V1187 are marked by *.
Generation and Expression of Mutant Factor H Proteins
Six recombinant mutant factor H proteins were expressed to assayand compare the role of six single amino acid substitutionson factor H ligand interaction. For exclusion of the contributionof the additional heparin and C3b binding sites, mutant proteinswere expressed using an SCR8 to SCR20 backbone. The recombinantmutant factor H proteins have single residue exchanges in SCR19(W1157R) or in SCR20 (W1183L, V1197A, R1210C, R1215G, or P1226S).The substitutions are positioned within or adjacent to the fourbinding regions identified from the spot analyses (Figure 1B).All purified mutant and wild-type factor H proteins showed comparablemobilities (Figure 2A).
Figure 2. Expression of recombinant mutant factor H proteins and heparin affinity chromatography. (A) Recombinant factor H proteins with the indicated aHUS-associated mutations were generated on a factor H 8-20 framework and expressed in insect cells. The mutant proteins were purified by nickel affinity chromatography, separated by SDS-PAGE, and identified by silver staining. (B) Heparin affinity chromatography of mutant factor H proteins. The proteins were loaded onto a heparin column, the matrix was washed thoroughly, and bound proteins were eluted using a linear NaCl gradient. The various fractions were analyzed by SDS-PAGE and Western blotting. As the elutions were performed under identical conditions, the elution profiles of the mutants can be compared directly.
Binding of Mutant Factor H Proteins to Heparin
Five mutant and the wild-type factor H SCR8-SCR20 proteins wereassayed using heparin affinity chromatography. Bound proteinswere eluted with a linear salt gradient, and the various elutefractions were collected and analyzed by SDS-PAGE and Westernblotting. All mutant proteins bound to the heparin matrix butwere eluted before the wild-type protein (Figure 2B). Wild-typefactor H SCR8-SCR20 was maximally eluted in fraction 34 at approximately340 mM NaCl. The W1157R mutant showed maximal elution in fraction32 (320 mM NaCl). The two mutant proteins with W1183L and V1197Ashowed very similar characteristics and were eluted in fractions29 and 28 (290 mM NaCl), whereas mutant R1210C was eluted infraction 30 (305 mM NaCl) and mutant P1226S was eluted in fraction27 (285 mM NaCl). These experiments show that single amino acidexchanges within SCR19 and SCR20 significantly weakened heparinbinding. The mutation in SCR19 (W1157R) caused a minor reductionin heparin affinity, whereas the other five in SCR20 had a largereffect.
Binding of Mutant Factor H Proteins to C3b and C3d
The C-terminus of factor H is central for interaction with complementcomponent C3. The C3 interaction was analyzed by surface plasmonresonance. C3b or C3d was coupled to the sensor chip, and thefive mutant and the wild-type factor H SCR8-SCR20 proteins wereinjected. The wild-type fragment showed prominent binding toC3b and C3d, as judged by the strong association and dissociationprofile (Figure 3, A and B). The five mutant proteins showedseverely reduced binding to immobilized C3b and C3d (Figure 3,A and B). A low but residual binding affinity was observedfor the mutant proteins W1183L and R1210C, whereas no interactionswith C3b or C3d was observed for the three mutant proteins W1157R,V1197A, and P1226S.
Figure 3. Binding of recombinant mutant factor H proteins to C3b and C3d. (A) Binding of mutant factor H (W1157R, W1183L, V1197A, R1210C, R1215G, and P1226S [dashed lines]) and wild-type factor H SCR8-SCR20 (solid line) to immobilized C3b was analyzed by surface plasmon resonance. Upon injection of the protein into the flow cell, both the association and the dissociation profiles were recorded. (B) The corresponding binding of recombinant mutant and wild-type factor H proteins to C3d was analyzed. (C) Binding of the mutant and wild-type factor H proteins to C3b and C3d using an ELISA approach. nd, not determined.
In addition, an ELISA test was used to assay C3 binding. C3bor C3d was immobilized to the surface of a microtiter plate,and the binding of six mutant factor H proteins was tested.The binding activities of the mutated proteins W1183L, V1197A,R1210C, and R1215G was reduced to approximately 50% of the wild-typeprotein. Binding of the mutant W1157R was strongly reduced,whereas that for the P1226S mutant was completely abolished(Figure 3C).
Binding to Endothelial Cells
The binding of five aHUS-associated mutant proteins to humanumbilical vein endothelial cells was assayed by confocal microscopy(Figure 4). Cells that were cultivated in serum-free mediumwere incubated with each protein and stained with factor H antiserum.The binding of factor H is indicated by a specific green fluorescencesignal. Again, all five mutant factor H proteins showed weakcell binding activity, in contrast to the wild-type factor HSCR8-SCR20 fragment, which showed prominent binding (Figure 4).
Figure 4. Binding of recombinant mutant and wild-type factor H proteins to human umbilical vein endothelial cells. Cells that were cultivated in serum-free medium were incubated with supernatant that contained either the indicated recombinant proteins W1157R (A), W1183L (B), V1197A (C), R1210C (D), and R1215G (E) or wild-type factor H SCR8-SCR20 (F). Cells were used directly for staining with factor H antiserum in combination with Alexa 488–labeled secondary antiserum (left). Cell surface structure was visualized with Alexa 633–labeled wheat germ agglutinin (right). Staining of the DNA with DAPI identifies the position of the nuclei. The bars in wild-type factor H show the length of 20 µm.
Localization of Peptides and Mutations in a Molecular Model
Molecular models for the SCR19 and SCR20 domains were generatedby homology modeling with two crystal structure models showingthe highest sequence identity with these domains (27). Eventhough the linker between SCR19 and SCR20 is short at threeresidues, the high variability in known inter-SCR orientations(29) meant that it was not possible to predict the relativearrangement of SCR19 and SCR20. Nonetheless, the mapping ofthe active regions from the peptide spot analysis showed thatall three regions in SCR20 were located in proximity to eachother at the C-terminal end of SCR20, which then is presumedto interact with all three of the heparin, C3b, and C3d ligands(Figure 5). The active region in SCR19 is located at the loopbetween -strands 4 and 5 and along -strand 5. If SCR19 and SCR20are orientated to form a V-shaped structure, then it is possiblethat the four regions may form a single binding surface acrossSCR19 and SCR20.
Figure 5. Position of the aHUS-associated mutations in SCR19 and SCR20 of factor H. (A) Sequence of SCR20 of factor H. The disulfide bridges that are formed with CysI-CysIII and CysII-CysIV connectivities are indicated. The residues within regions II, III, and IV involved in heparin binding are shown in green, and the aHUS-associated mutations of this study are highlighted in red. Amino acids that are conserved in all factor H proteins are shown in gray. (B) Homology models of SCR19 and SCR20 to indicate the location of regions I to IV and the six aHUS mutations. The type A W1183L, R1210C, and R1215G mutants (red) are positioned within the heparin binding site at the C-terminal tip of SCR20 and may be proximate to the type A mutant (red) in SCR19 (W1157R). The type B V1197A and P1226S mutants (light red) are buried within the SCR20 structure. The top panel shows a space-filling view of SCR19 and SCR20, whereas the bottom panel shows the same models in a ribbon view.
The SCR19 and SCR20 models were also used to interpret positionof the individual mutant residues. On the basis of the accessibilityof the various sidechains, the mutations can be subdivided.Type A corresponds to surface-exposed residues (W1157R, W1183L,R1210C, and R1215G), whereas type B corresponds to residueswhose side chains are buried within the SCR domain (V1197A andP1226S). It should be noted that SCR20 is atypical of SCR domainsin that the conserved Trp residue is adjacent to the third Cysresidue of SCR20 and not spaced by four residues (26), and thismay affect the predicted sidechain accessibility of R1210C andR1215G. Although the accessibilities are tentative pending anexperimental determination of the SCR19 and SCR20 structures,the alignment of V1197A with -strand 3 in SCR20 and that ofP1226S close to the buried C-terminal Cys residue add confidenceto the buried accessibility assignments of these two sidechains.Both of the type B mutations show the largest effects when assayedby heparin affinity chromatography (Figure 2B), and likewiseon the binding of C3b or C3d when followed by surface plasmonresonance or ELISA analyses (Figure 3).
The largest group of aHUS-associated mutations known to dateoccur in the gene coding for the human immune regulator factorH, and >60% of these reported mutations are clustered withinthe C-terminal recognition region in SCR19 and SCR20 (1,2,26).Our study shows that at least six of these mutations are relateddirectly with the pathogenesis of aHUS. Six mutant factor Hproteins that were reported for patients with aHUS were expressedon a SCR8-SCR20 backbone, and all mutant proteins showed defectiveligand binding and endothelial cell interaction (Table 1). Bypeptide spot analyses, we identified four linear binding regionswithin SCR19 and SCR20 that mediate binding to heparin, C3b,and C3d. Molecular modeling satisfactorily accounted for theidentification of four peptide regions and the six aHUS mutationsin terms of a possible common surface in SCR19 and SCR20 thatinteracts with heparin, C3b, and C3d. The similar functionalconsequences of the six aHUS-associated mutants of factor Hsuggest a common mechanism for the pathophysiology of this disease:Upon an inflammatory insult, the defective cell recognitionof factor H reduces its regulatory activities at the surfaceof endothelial cells, which results in cell damage and exposureof the subendothelial matrix.
Table 1. Summary of binding characteristics of the six mutant factor H proteins to three ligands (heparin, C3b, and C3d) and to endothelial cells
By peptide spot analysis, we identified four linear bindingregions in the recognition domain of factor H that mediate ligandinteraction, and the same or very similar four regions mediateheparin, C3b, and C3d binding. Region I is located in SCR19and is 10 residues long (residues 1145 to 1154). SCR20 includesthree binding regions, of which region II is 10 residues long(residues 1178 to 1187), region III is 14 residues long (residues1202 to 1215), and region IV at the C-terminus is three residueslong (residues 1229 to 1231; Figure 1B). The peptide spot analysisshowed that C3b interaction is located mainly in SCR20 and toa great extent overlaps with the heparin binding region (Figure 1A).This result agrees with previous reports that showed competitionof C3b binding of a factor H SCR15 to SCR20 fragment with heparin(29). The identification of these four regions accounts fordiscrepancies in three previous studies. Theoretical homologymodeling based on 12 aHUS mutations had predicted that fourbasic residues (R1182, K1186, K1188, and R1192) at the interfacebetween SCR19 and SCR20 may interact with heparin (30). Ourwork shows that the first two of these mutated residues arelocated in region II, and the other two follow immediately (Figure 1B).This is the first experimental evidence to support thisprediction. A separate theoretical homology model suggestedthat K1108 in SCR19 and K1202, R1206, R1210, K1222, K1230, andR1231 in SCR20 interact with heparin (29). K1108 is far fromregions I to IV, and its predicted role is not supported bythe peptide assay, whereas the role of the remaining six residueslocated within region III (K1202, R1206, and R1210) or regionIV (K1230 and R1231) or between them (K1222) is experimentallysupported. A recombinant factor H SCR15-SCR20 fragment withfive mutations based on three residues in region III (R1203E,R1206E, and R1210S) and two residues in region IV (K1230S andR1231A) did not bind to heparin (29). Hence, the peptide spotanalyses are in agreement with a second set of heparin bindingresidues in SCR20. In a third study, the crystal structure ofthe complex between decameric heparin and SCR4 of VCP was reported(31). An overlay of the heparin binding residues of SCR4 inVCP with a model of SCR20 in human factor H shows that the sevenVCP heparin binding residues correspond to five factor H residuesin region III (K1202, R1203, R1206, R1210, and R1215) and twoFactor H residues in region IV (K1230 and R1231). The peptidespot analyses are consistent with the comparison made with theheparin-VCP crystal structure.
The involvement of the aHUS-associated mutations with the functionalproperties of factor H was supported by the six mutants of factorH. Clinically, all six mutant proteins are expressed and secreted(2). The mutants showed perturbation of heparin binding (weakestto strongest: W1157R, R1210C, W1183L, V1197A, and P1226S; Figure 2B)and a strong reduction in C3b binding (W1183L, V1197A, R1215G,R1210C, W1157R, and P1226S; weakest to strongest; Figure 3C)and C3d (V1197A, R1210C, W1183L, R1215G, W1157R, and P1226S;again weakest to strongest; Figure 3C). It is interesting thateven though factor H molecules with mutations W1183L, V1197A,and R1210C isolated from patient sera had C3b binding sitesin the amino terminal ends of factor H, these mutant proteinsalso showed severe reduction in binding to immobilized C3b (32).These data demonstrate that the C-terminal SCR domains of factorH form a major binding site for C3b. Whereas heparin and C3bbinding is concentrated in SCR20, the binding of C3d is mappedto SCR19 and SCR20 (Figure 2) and is overlapping with the heparinbinding sites. Although no binding of factor H fragment SCR15to SCR19 to immobilized C3d was demonstrated (29), SCR19 issupposed to be involved in C3d binding. This is underlined bydata showing that mutation W1157R in SCR19 has a significanteffect on C3b and C3d binding, but this effect is much reducedfor heparin binding (Figures 2B and 4A). Both density and distributionof surface-deposited C3b affect the affinity of factor H toa cell surface. Thus, it seems that the combination of defectiveheparin and C3b binding contributes to a defective local complementcontrol at the cell surface.
The homology modeling of SCR19 and SCR20 provided further insightinto recognition function of the four identified binding regionsand the six mutated residues. All four linear binding regionsthat were identified by peptide spot analyses are surface exposedand accessible to ligands. The SCR20 structure shows that regionsII, III, and IV are spatially brought together at the C-terminaltip of SCR20 (Figure 5B). This is consistent with the proposedanalogous binding of heparin to the C-terminal tip of VCP inthe heparin-VCP crystal structure (31). In SCR19, region I canbe brought into proximity with regions II, III, and IV to forma single contiguous surface. This indicates a role of both SCR19and SCR20 in binding to its three ligands and is consistentwith an arrangement of the SCR19 and SCR20 in the four best-fitmodels for intact factor H (33). The defective binding of thesix mutant factor H proteins is most easily explained by differentaccessibility of individual residues within the SCR domain.Type A residues are accessible, and type B residues are buried(<20% solvent accessibility). In terms of mutations, it isof interest that the two type B mutations V1197A and P1226Sshowed the largest effect on heparin binding; in addition, P1226Shad the largest effect on C3b and C3d binding. The change froma bulky aliphatic sidechain to a small one (V1197A) or thatfrom a hydrophobic amino acid to a small hydrophilic amino acid(P1226S) has a disproportionate effect on factor H activitiescompared with the type A mutations. It is of interest to seewhether functionally active SCR domains are generally sensitiveto alterations in their buried residues that can distort thefolding of the SCR domain.
So far, 36 of the reported aHUS-associated factor H gene mutationsare located in SCR19 and SCR20. By this study, mutations andamino acid substitutions in the two SCR domains disturb theoverall recognition function of this region. In consequencemutant factor H cannot bind properly to the endothelial cellsand thus cannot inhibit complement activation. This is particularlytrue during inflammation, when the alternative complement pathwayis highly induced and the endothelium needs maximal protection.In this scenario, presumably thrombi that cannot be clearedproperly are formed in the kidney, causing kidney damage overthe time.
Acknowledgments
This work was funded by the Deutsche Forschungsgemeinschaftand the KIDNEEDS Foundation (Cedar Rapids, Iowa). S.J.P. andR.E.S. are supported by the Wellcome Trust and the Medical ResearchCouncil.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-strands 
