Complement Control Protein Domains Research Articles

Human Factor H Interacts Selectively with Neisseria gonorrhoeae and Results in Species-Specific Complement Evasion

Complement forms a key arm of innate immune defenses against gonococcal infection. Sialylation of gonococcal lipo-oligosaccharide, or expression of porin 1A (Por1A) protein, enables Neisseria gonorrhoeae to bind the alternative pathway complement inhibitor, factor H (fH), and evade killing by human complement. Using recombinant fH fragment-murine Fc fusion proteins, we localized two N. gonorrhoeae Por1A-binding regions in fH: one in complement control protein domain 6, the other in complement control proteins 18-20. The latter is similar to that reported previously for sialylated Por1B gonococci. Upon incubation with human serum, Por1A and sialylated Por1B strains bound full-length human fH (HufH) and fH-related protein 1. In addition, Por1A strains bound fH-like protein 1 weakly. Only HufH, but not fH from other primates, bound directly to gonococci. Consistent with direct HufH binding, unsialylated Por1A gonococci resisted killing only by human complement, but not complement from other primates, rodents or lagomorphs; adding HufH to these heterologous sera restored serum resistance. Lipo-oligosaccharide sialylation of N. gonorrhoeae resulted in classical pathway regulation as evidenced by decreased C4 binding in human, chimpanzee, and rhesus serum but was accompanied by serum resistance only in human and chimpanzee serum. Direct-binding specificity of HufH only to gonococci that prevents serum killing is restricted to humans and may in part explain species-specific restriction of natural gonococcal infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates.

Antibodies against Kaposi sarcoma-associated herpes virus (KSHV) complement control protein (KCP) in infected individuals

Kaposi sarcoma-associated herpesvirus (KSHV) is the most important etiopathological factor of Kaposi's sarcoma (KS) and some specific types of malignant lymphomas. One of the viral lytic genes encodes the KSHV complement control protein (KCP), which functionally mimics human complement inhibitors. Although this protein provides an advantage for evading the complement attack, it can serve as target for adaptive immune response. Herein, we identified anti-KCP IgG antibodies in patients with KS and KSHV-related lymphomas. KCP-specific antibodies were only detected in sera of those patients who had high titres of antibodies against lytic or latent KSHV antigens. Complement control protein domain 2 (CCP2) was found to be the most immunogenic part of the KCP protein. Furthermore, pre-incubation of KCP-expressing CHO cells with patient sera containing anti-KCP antibodies resulted in an increased complement deposition when incubated with human serum.

Separation of decay‐accelerating and cofactor functional activities of Kaposi's sarcoma‐associated herpesvirus complement control protein using monoclonal antibodies

Complement is an essential part of the innate immune system, which clears pathogens without requirement for previous exposure, although it also greatly enhances the efficacy and response of the cellular and humoral immune systems. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently identified human herpesvirus and the likely aetiological agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. We previously reported that the KSHV complement control protein (KCP) was expressed on infected cells and virions, and could inhibit complement through decay-accelerating activity (DAA) of the classical C3 convertase and cofactor activity (CFA) for factor I (FI)-mediated degradation of C4b and C3b, as well as acting as an attachment factor for binding to heparan sulphate on permissive cells. Here, we determined the ability of a panel of monoclonal anti-KCP antibodies to block KCP functions relative to their recognized epitopes, as determined through binding to recombinant KCP containing large (entire domain) or small (2-3 amino acid residue) alterations. One antibody recognizing complement control protein (CCP) domain 1 blocked heparin binding, DAA and C4b CFA, but was poor at blocking C3b CFA, while a second antibody recognizing CCP4 blocked C3b CFA and 80% DAA, but not C4b CFA or heparan sulphate binding. Two antibodies recognizing CCP2 and CCP3 were capable of blocking C3b and C4b CFA and heparan sulphate binding, but only one could inhibit DAA. These results show that, while KCP is a multifunctional protein, these activities do not completely overlap and can be isolated through incubation with monoclonal antibodies.

Molecular Characterization of the Interaction between Porins of Neisseria gonorrhoeae and C4b-Binding Protein

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.

Three new high‐prevalence antigens in the Cromer blood group system

The Cromer blood group system consists of nine high-prevalence and three low-prevalence antigens carried on decay-accelerating factor (DAF). This report describes three new Cromer high-prevalence antigens, named ZENA, CROV, and CRAM. Sequence analyses were performed on DNA from three probands whose serum samples each contained an alloantibody to a high-prevalence antigen in the Cromer blood group system. Polymerase chain reaction-restriction fragment length polymorphism analysis to detect the mutation encoding the CROV- phenotype was performed on 100 Croatian donors. To map the respective epitopes, DAF deletion mutants were tested by immunoblotting with eluates containing the antibodies. In each proband, sequence analysis revealed a single-nucleotide substitution in DAF: ZENA, 726T>G mutation, predicted change His242Gln; CROV, 466G>A mutation, predicted change Glu156Lys; and CRAM, 740A>G mutation, predicted change Gln247Arg. By analysis of DAF deletion mutants, the CROV antigenic determinant mapped to the complement control protein (CCP) domain 2, which is encoded by exon 3, whereas ZENA and CRAM mapped to CCP4, which is encoded by exon 6. This study describes three novel high-prevalence antigens in the Cromer blood group system each characterized by a predicted single-amino-acid substitution. The antigens have been assigned the following International Society of Blood Transfusion (ISBT) numbers: ZENA is CROM13, CROV is CROM14, and CRAM is CROM15.

Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.

A Novel Mannose-binding Lectin-associated Serine Protease 1/3 Gene Variant

The ficolins and mannose-binding lectin (MBL) create complexes with three different serine proteases (MASP-1, MASP-2 and MASP-3) and a truncated non-enzymatic form of MASP-2 (sMAP). MASP-2 is able to activate complement by cleavage of C4 and C2, while the physiological functions of MASP-1, MASP-3 and sMAP still are debated. MASP-1 and MASP-3 are alternative spliced forms of the same MASP gene. To gain insight in the molecular variation in the MASP-1/3 gene, we undertook a systematic study of the protein coding sequences of the MASP-1/3 gene. The coding regions of the MASP-1/3 gene were sequenced in 92 healthy Caucasian donors. A total of six nucleotide substitutions were detected. Five were detected only once. One polymorphism identified in exon 10 at position +50074 (rs 38343199) relative to the transcription start site resulting in the amino acid substitution of a glycine (GGG) with a glutamic acid residue (GAG) in the second complement control protein domain was observed. The frequency of this allele in 305 blood donors, 90 patients with systemic lupus erythematosus and 234 patients with the systemic inflammatory response syndrome (SIRS) and/or sepsis was 0.03, 0.017 and 0.03 respectively. No significant differences in genotype frequencies between the groups were observed (P > 0.45). However, the SIRS/sepsis group deviated from the Hardy-Weinberg expectations due to one variant allele homozygote (P = 0.07), which was not observed in the other groups. In conclusion, the MASP1/3 gene harbours a low-frequent polymorphic site resulting in an amino acid substitution, which may influence the function of the gene product.

The Factor H Variant Associated with Age-related Macular Degeneration (His-384) and the Non-disease-associated Form Bind Differentially to C-reactive Protein, Fibromodulin, DNA, and Necrotic Cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Crystal Structure and Mutational Analysis of the DaaE Adhesin of Escherichia coli

DaaE is a member of the Dr adhesin family of Escherichia coli, members of which are associated with diarrhea and urinary tract infections. A receptor for Dr adhesins is the cell surface protein, decay-accelerating factor (DAF). We have carried out a functional analysis of Dr adhesins, as well as mutagenesis and crystallographic studies of DaaE, to obtain detailed molecular information about interactions of Dr adhesins with their receptors. The crystal structure of DaaE has been solved at 1.48 A resolution. Trimers of the protein are found in the crystal, as has been the case for other Dr adhesins. Naturally occurring variants and directed mutations in DaaE have been generated and analyzed for their ability to bind DAF. Mapping of the mutation sites onto the DaaE molecular structure shows that several of them contribute to a contiguous surface that is likely the primary DAF-binding site. The DAF-binding properties of purified fimbriae and adhesin proteins from mutants and variants correlated with the ability of bacteria expressing these proteins to bind to human epithelial cells in culture. DaaE, DraE, AfaE-III, and AfaE-V interact with complement control protein (CCP) domains 2-4 of DAF, and analysis of the ionic strength dependence of their binding indicates that the intermolecular interactions are highly electrostatic in nature. The adhesins AfaE-I and NfaE-2 bind to CCP-3 and CCP-4 of DAF, and electrostatic interactions contribute significantly less to these interactions. These observations are consistent with structural predictions for these Dr variants and also suggest a role for the positively charged region linking CCP-2 and CCP-3 of DAF in electrostatic Dr adhesin-DAF interactions.

The Role of Decay-accelerating Factor as a Receptor for Helicobacter pylori and a Mediator of Gastric Inflammation

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Phenotypic expression of factor H mutations in patients with atypical hemolytic uremic syndrome

We investigated the phenotypic expression of factor H mutations in two patients with atypical hemolytic uremic syndrome (HUS). Factor H in serum was assayed by rocket immunoelectrophoresis, immunoblotting, and double immunodiffusion and in tissue by immunohistochemistry. Functional activity was analyzed by hemolysis of sheep erythrocytes and binding to endothelial cells. A homozygous mutation in complement control protein (CCP) domain 10 of factor H was identified in an adult man who first developed membranoproliferative glomerulonephritis and later HUS. C3 levels were very low. The patient had undetectable factor H levels in serum and a weak factor H 150 kDa band. Double immunodiffusion showed partial antigenic identity with factor H in normal serum owing to the presence of factor H-like protein 1. Strong specific labeling for factor H was detected in glomerular endothelium, mesangium and in glomerular and tubular epithelium as well as in bone marrow cells. A heterozygous mutation in CCP 20 of factor H was found in a girl with HUS. C3 levels were moderately decreased at onset. Factor H levels were normal and a normal 150 kDa band was present. Double immunodiffusion showed antigenic identity with normal factor H. Factor H labeling was minimal in the renal cortex. Factor H dysfunction was demonstrated by increased sheep erythrocyte hemolysis and decreased binding to endothelial cells. In summary, two different factor H mutations associated with HUS were examined: in one, factor H accumulated in cells, and in the other, membrane binding was reduced.

Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp‐mediated C3b and C4b degradation

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood-brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+ E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA- E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+ E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum.

The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1–2

The Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP) inhibits the human complement system, and is similar in structure and function to endogenous complement inhibitors. Other inhibitors such as C4b-binding protein and factor H, as well as the viral homologue vaccinia virus complement control protein are known to bind heparin and, for the two latter, also to glycosaminoglycans at the surface of cells. We report here that KCP also binds to heparin at physiological ionic strength. With help of site directed mutagenesis, positively charged amino acids in the two N-terminal complement control protein (CCP) domains 1–2 were found to be necessary for heparin binding. In silico molecular docking of heparin to KCP confirmed the experimental data, and further explored the heparin binding site, enabling us to present a model of the KCP–heparin interaction. Furthermore, the docking analysis also yielded insights of the KCP structure, by indicating that the angle between CCP domains 1–2 during the initial binding of heparin is more extended than in the model we have previously presented. We also found that KCP binds to heparan sulfate and weakly to glycosaminoglycans at the surface of cells. This might indicate that KCP at the surface of viral particles aids in the primary attachment to the target cells, which is known to involve binding to heparan sulfate. Therefore, the present study contributes to the knowledge of heparin–protein interactions in general as well as to the understanding of the biology of KSHV.

Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein

Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.

FHR-4A: a new factor H-related protein is encoded by the human FHR-4 gene

We describe a new member of the human Factor H protein family, termed Factor H-related protein 4A (FHR-4A). The corresponding cDNA sequence was isolated and encodes a secreted protein of 559 amino acids, with a predicted molecular weight of 63.2 kDa. Apparently, this novel cDNA is derived from the human FHR-4 gene. Genetic analysis shows that the human FHR-4 gene is composed of 10 coding exons, and two distinct mRNA transcripts are derived from this gene by alternative splicing. The short FHR-4B form represents a truncated variant and encodes a secreted protein of five domains (previously termed FHR-4). The long transcript encodes the novel FHR-4A protein that is composed of nine complement control protein (CCP) domains. A unique feature of FHR-4A is the tandem arrangement of four CCP domains forming a 'natural dimer' of the short isoform. The FHR-4A protein is identified in human plasma as a 86 kDa protein. The difference between the predicted and observed molecular masses is explained by glycosylation. Comparison of the deduced protein sequence of FHR-4A with peptides from a 86 kDa apolipoprotein described by us earlier suggests that the long form, FHR-4A, represents this apoprotein. In summary, FHR-4A is a new Factor H-related protein with a unique domain composition, that is, an internal duplication of four CCP domains. To our knowledge, FHR-4A provides the first evidence for alternative splicing among Factor H-related genes.

The Hyphal and Yeast Forms ofCandida albicansBind the Complement Regulator C4b-Binding Protein

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

The Kaposi's Sarcoma-associated Herpesvirus Complement Control Protein Mimics Human Molecular Mechanisms for Inhibition of the Complement System

Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O. B., Blackbourn, D. J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.

Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H.

The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30 degrees C-90 degrees C. We found that predominantly beta-sheet structure of both proteins was stable up to 70 degrees C, and that a reversible conformational change toward alpha-helix was apparent at temperatures ranging from 70 degrees C to 90 degrees C. The ability of both proteins to inhibit complement was not impaired after incubation at 95 degrees C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in beta-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.