Mapping of regions within the vaccinia virus complement control protein involved in dose-dependent binding to key complement components and heparin using surface plasmon resonance

The vaccinia virus complement control protein (VCP) is involved in modulating the host inflammatory response by blocking both pathways of complement activity through its ability to bind C3b and C4b. Other activities arise from VCP's ability to strongly bind heparin. To map regions within VCP involved in binding complement and heparin experimentally, surface plasmon resonance (SPR) and recombinantly expressed VCP (rVCP) constructs were employed. Using C3b or heparin as the immobilized ligand, various rVCP constructs were tested for their ability to bind. Results suggest that VCP is the smallest functional unit able to bind C3b, thereby blocking complement activity, and only a single site, the large basic region near the C-terminus, is involved in heparin binding. Kinetic analysis was also performed to determine the relative binding affinities between rVCP and complement (C3-MA and C4b), as well as rVCP and heparin. rVCP was found to possess a significantly greater affinity for C3-MA than C4b, as indicated by the 1.50e3-fold greater association rate constant (k(a)). This study provides insights for the design of new therapeutic proteins capable of blocking complement activation.     

Vaccinia virus complement control protein is monomeric,and retains structural and functional integrity after exposure to adverse conditions

Vaccinia virus complement control protein (VCP) possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. Recently, the structure of the complete molecule of VCP was determined by X-ray crystallography. Two or three VCP molecules were packed within the unit cells of both crystal forms. Using gel filtration, VCP has now been shown to exist as a monomer in solution. To test the stability of this molecule, VCP was studied by nuclear magnetic resonance (NMR) over a range of temperatures and by differential scanning calorimetry (DSC). It was also subjected to adverse physical conditions, including, freeze-thawing, changes in pH, changes in temperature, and storage at room temperature. VCP melts fully reversibly, and it maintained its 3-D structure and the ability to inhibit serum-induced hemolysis of sheep red blood cells after exposure to many extreme conditions. The robustness of VCP may be rationalized in terms of its architecture.     

Vaccinia virus complement control protein is monomeric,and retains structural and functional integrity after exposure to adverse conditions

Vaccinia virus complement control protein (VCP) possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. Recently, the structure of the complete molecule of VCP was determined by X-ray crystallography. Two or three VCP molecules were packed within the unit cells of both crystal forms. Using gel filtration, VCP has now been shown to exist as a monomer in solution. To test the stability of this molecule, VCP was studied by nuclear magnetic resonance (NMR) over a range of temperatures and by differential scanning calorimetry (DSC). It was also subjected to adverse physical conditions, including, freeze–thawing, changes in pH, changes in temperature, and storage at room temperature. VCP melts fully reversibly, and it maintained its 3-D structure and the ability to inhibit serum-induced hemolysis of sheep red blood cells after exposure to many extreme conditions. The robustness of VCP may be rationalized in terms of its architecture.     

Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans

Vaccinia virus complement control protein (VCP) inhibits both pathways of complement activation through binding the third and fourth components. A homolog of mammalian regulators of complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. This, the structure of any intact regulator of complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of complement.     

Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.     

Influence of glycosylation and oligomerization of vaccinia virus complement control protein on level and pattern of functional activity and immunogenicity

Vaccinia virus complement control protein (VCP) is one of the proteins encoded by vaccinia virus to modulate the host inflammatory response. VCP modulates the inflammatory response and protects viral habitat by inhibiting the classical and the alternative pathways of complement activation. The extended structure of VCP, mobility between its sequential domains, charge distribution and type of residues at the binding regions are factors that have been identified to influence its ability to bind to complement proteins. We report that a Lister strain of vaccinia virus encodes a VCP homolog (Lis VCP) that is functional, glycosylated, has two amino acids less than the well-characterized VCP from vaccinia virus WR strain (WR VCP), and the human smallpox inhibitor of complement enzymes (SPICE) from variola virus. The glycosylated VCP of Lister is immunogenic in contrast to the weak immunogenicity of the nonglycosylated VCP. Lis VCP is the only orthopoxviral VCP homolog found to be glycosylated, and we speculate that glycosylation influences its pattern of complement inhibition. We also correlate dimerization of VCP observed only in mammalian and baculovirus expression systems to higher levels of activity than monomers, observed in the yeast expression system.     

The HIV-1 envelope glycoprotein gp120 features four heparan sulfate binding domains, including the co-receptor binding site

It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg(419), Lys(421), and Lys(432), which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition.     

Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein

The vaccinia virus complement control protein (VCP) is secreted by infected cells and has been shown to inhibit complement activation through interactions with C3b/C4b. It contains four short consensus repeat (SCR) domains. It has been suggested that all four SCRs are required for VCP's activity. To elucidate which SCR domains are involved in abolishing complement-enhanced neutralization of vaccinia virus virions, we generated and characterized a panel of mouse monoclonal antibodies (MAbs) raised against VCP. Ten MAbs were isolated and all recognized VCP on Western blots under reducing conditions as well as native-bound VCP in a sandwich enzyme-linked immunosorbent assay. Three of the 10 MAbs (2E5, 3D1, and 3F11) inhibited VCP's abolition of complement-enhanced neutralization of vaccinia virus virions. These MAbs blocked the interaction of VCP with C3b/C4b. The seven remaining MAbs did not alter VCP function in the complement neutralization assay and recognized VCP bound to C3b/C4b. To understand MAb specificity and mode of interaction with VCP, we mapped the MAb binding regions on VCP. The seven nonblocking MAbs all bound to the first SCR of VCP. One of the blocking MAbs recognized SCR 2 while the other two recognized either SCR 4 or the junction between SCRs 3 and 4, indicating that structural elements involved in the interaction of VCP with C3b/C4b are located within SCR domains 2 and 3 and 4. These anti-VCP MAbs may have clinical significance as therapeutic inhibitors of VCP's complement control activity and may also offer a novel approach to managing vaccinia virus vaccine complications that occur from smallpox vaccination.     

Vaccinia complement control protein: Multi-functional protein and a potential wonder drug

Vaccinia virus complement control protein (VCP) was one of the first viral molecules demonstrated to have a role in blocking complement and hence in the evasion of host defense. Structurally it is very similar to the human C4b-BP and the other members of complement control protein. Functionally it is most similar to the CR1 protein. VCP blocks both major pathways of complement activation. The crystal structure of VCP was determined a little over a year ago and it is the only known structure of an intact and complete complement control protein. In addition to binding complement, VCP also binds to heparin. These two binding abilities can take place simultaneously and contribute to its many function and to its potential use in several inflammatory diseases, e.g. Alzheimer's disease (AD), CNS injury, xenotransplantation, etc. making it a truly fascinating molecule and potential drug.     

Potential mechanisms for the regulation of growth factor binding by heparin

Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG.     

Streptococcal beta protein has separate binding sites for human factor H and IgA-Fc.

The group B streptococcus (GBS) is the most important cause of life-threatening bacterial infections in newborn infants. Protective immunity to GBS infection is elicited by several surface proteins, one of which, the beta protein, is known to bind human IgA-Fc. Here, we show that the beta protein also binds human factor H (FH), a negative regulator of complement activation. Absorption experiments with whole human plasma demonstrated binding of FH to a GBS strain expressing beta protein but not to an isogenic beta-negative mutant. This binding was due to a direct interaction between beta and FH, as shown by experiments with purified proteins. Inhibition tests and studies with beta fragments demonstrated that FH and IgA-Fc bind to separate and nonoverlapping regions in beta. Heparin, a known ligand for FH, specifically inhibited the binding between beta and FH, suggesting that FH has overlapping binding sites for beta and heparin. Bacteria-bound FH retained its complement regulatory activity, implying that beta-expressing GBS may use bound FH to evade complement attack. The finding that beta protein binds FH adds to a growing list of interactions between human pathogens and complement regulatory proteins, supporting the notion that these interactions are of general importance in bacterial pathogenesis.     

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.     

Cell surface expression of the vaccinia virus complement control protein is mediated by interaction with the viral A56 protein and protects infected cells from complement attack

The vaccinia virus (VACV) complement control protein (VCP) is the major protein secreted from VACV-infected cells. It has been reported that VCP binds to the surfaces of uninfected cells by interacting with heparan sulfate proteoglycans (HSPGs). In this study, we show that VCP is also expressed on the surfaces of infected cells and demonstrate that surface localization occurs independently of HSPGs. Since VCP does not contain a transmembrane domain, we hypothesized that VCP interacts with a membrane protein that localizes to the infected-cell surface. We show that the VACV A56 membrane protein is necessary for the cell surface expression of VCP and demonstrate that VCP and A56 interact in VACV-infected cells. Since the surface expression of VCP was abrogated by reducing agents, we examined the contribution of an unpaired cysteine residue on VCP to VCP surface expression and VCP's interaction with A56. To do this, we mutated the unpaired cysteine in VCP and generated a recombinant virus expressing the altered form of VCP. Following the infection of cells with the mutant virus, VCP was neither expressed on the cell surface nor able to interact with A56. Importantly, the cell surface expression of VCP was found to protect infected cells from complement-mediated lysis. Our findings suggest a new function for VCP that may be important for poxvirus pathogenesis and impact immune responses to VACV-based vaccines.     

Transforming growth factor-beta 1 is a heparin-binding protein: identification of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-beta 1.

Previous studies indicated that a major factor in heparin's ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor-beta 1 (TGF-beta 1). Heparin appeared to bind directly to TGF-beta 1 and to prevent the association of TGF-beta 1 with alpha 2-macroglobulin (alpha 2-M). The present studies indicate that 20-70% of iodinated TGF-beta 1 binds to heparin-Sepharose and the retained fraction is eluted with approximately 0.37 M NaCl. Native, unlabelled platelet TGF-beta 1, however, is completely retained by heparin-Sepharose and eluted with 0.9-1.2 M NaCl. Using synthetic peptides, the regions of TGF-beta 1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF-beta 1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin-binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF-beta 1, were found to bind to heparin-Sepharose and were eluted with 0.15 M-0.30 M NaCl. Only two of these regions were capable of blocking the binding of heparin to 125I-TGF-beta. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF-beta 1. The ability of TGF-beta 1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF-beta 1 may be a factor in some of the diverse biological actions of heparin.     

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.     

Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is approximately 90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.     

Solution structure and dynamics of the central CCP module pair of a poxvirus complement control protein

The complement control protein (CCP) module (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CCP modules form specific binding sites for other molecules. Hence the orientation in space of a CCP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. Vaccinia virus complement control protein (VCP) is a complement regulatory protein composed of four tandemly arranged CCP modules. The solution structure of the carboxy-terminal half of this protein (CCP modules 3 and 4) has been solved previously. The structure of the central portion (modules 2 and 3, VCP approximately 2,3) has now also been solved using NMR spectroscopy at 37 degrees C. In addition, the backbone dynamics of VCP approximately 2,3 have been characterised by analysis of its (15)N relaxation parameters. Module 2 has a typical CCP module structure while module 3 in the context of VCP approximately 2,3 has some modest but significant differences in structure and dynamics to module 3 within the 3,4 pair. Modules 2 and 3 do not share an extensive interface, unlike modules 3 and 4. Only two possible NOEs were identified between the bodies of the modules, but a total of 40 NOEs between the short intermodular linker of VCP approximately 2,3 and the bodies of the two modules determines a preferred, elongated, orientation of the two modules in the calculated structures. The anisotropy of rotational diffusion has been characterised from (15)N relaxation data, and this indicates that the time-averaged structure is more compact than suggested by (1)H-(1)H NOEs. The data are consistent with the presence of many intermodular orientations, some of which are kinked, undergoing interconversion on a 10(-8)-10(-6) second time-scale. A reconstructed representation of modules 2-4 allows visualisation of the spatial arrangement of the 11 substitutions that occur in the more potent complement inhibitor from Variola (small pox) virus.     

Neurite growth-promoting protein (amphoterin, p30) binds syndecan.

A new ligand for syndecan (a cell surface heparan sulfate-rich proteoglycan) has been discovered. In the solid-phase binding assay utilizing small nitrocellulose discs to immobilize matrix molecules, binding of syndecan to neurite growth-promoting protein, p30/amphoterin, was observed. This binding was strongly dependent on the concentration of amphoterin used to coat the discs, but was saturable with an excess amount of syndecan. The interaction was inhibitable with heparan sulfate and heparin but less effectively with chondroitin sulfate, indicating that heparan sulfate chains of syndecan were involved in the binding. Anti-amphoterin antibodies inhibited the binding partially. Mouse mammary epithelial cells were shown to bind amphoterin directly but not after trypsin treatment or in the presence of heparin and to produce amphoterin in the extracellular space. Both syndecan and amphoterin were found to localize on lateral surfaces of newly adhered mammary epithelial cells. Toward confluency amphoterin amounts decreased. Because amphoterin can be localized to the same sites with syndecan and because of their interaction, amphoterin is a new putative pericellular ligand for syndecan. These interactions may be involved in the regulation of cell behavior.     

The amino-terminal part of PRELP binds to heparin and heparan sulfate

PRELP (proline, arginine-rich end leucine-rich repeat protein) is an extracellular matrix leucine-rich repeat protein. The amino-terminal region of PRELP differs from that of other leucine-rich repeat proteins in containing a high number of proline and arginine residues. The clustered proline and basic residues are conserved in rat, bovine, and human PRELP. Although the function of PRELP is not yet known, the clustered arginine residues suggest a heparan sulfate/heparin-binding capacity. We show here that PRELP indeed binds heparin and heparan sulfate. Truncated PRELP without the amino-terminal region does not bind heparin. The dissociation constant for the interaction of PRELP with heparin was determined by an in solution binding assay and by surface plasmon resonance analysis to be in the range of 10-30 nm. A 6-mer heparin oligosaccharide was the smallest size showing binding to PRELP. The binding increased with increasing length up to an 18-mer and depended on the degree of sulfation of heparin as well as heparan sulfate. Sulfate groups at all positions were shown to be of importance for the binding. Fibroblasts bind PRELP, and this interaction is inhibited with heparin, suggesting a function for PRELP as a linker between the matrix and cell surface proteoglycans.     

A new map of glycosaminoglycan and C3b binding sites on factor H

Human complement factor H, consisting of 20 complement control protein (CCP) modules, is an abundant plasma glycoprotein. It prevents C3b amplification on self surfaces bearing certain polyanionic carbohydrates, while complement activation progresses on most other, mainly foreign, surfaces. Herein, locations of binding sites for polyanions and C3b are reexamined rigorously by overexpressing factor H segments, structural validation, and binding assays. As anticipated, constructs corresponding to CCPs 7-8 and 19-20 bind well in heparin-affinity chromatography. However, CCPs 8-9, previously reported to bind glycosaminoglycans, bind neither to heparin resin nor to heparin fragments in gel-mobility shift assays. Introduction of nonnative residues N-terminal to a construct containing CCPs 8-9, identical to those in proteins used in the previous report, converted this module pair to an artificially heparin-binding one. The module pair CCPs 12-13 does not bind heparin appreciably, notwithstanding previous suggestions to the contrary. We further checked CCPs 10-12, 11-14, 13-15, 10-15, and 8-15 for ability to bind heparin but found very low affinity or none. As expected, constructs corresponding to CCPs 1-4 and 19-20 bind C3b amine coupled to a CM5 chip (K(d)s of 14 and 3.5 microM, respectively) or a C1 chip (K(d)s of 10 and 4.5 microM, respectively). Constructs CCPs 7-8 and 6-8 exhibit measurable affinities for C3b according to surface plasmon resonance, although they are weak compared with CCPs 19-20. Contrary to expectations, none of several constructs encompassing modules from CCP 9 to 15 exhibited significant C3b binding in this assay. Thus, we propose a new functional map of factor H.