Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.     

Comparison of binding characteristics of factors B and H to C3b on normal and paroxysmal nocturnal hemoglobinuria erythrocytes

To determine if aberrant interactions of the endogenous control proteins with cell-bound C3b contribute to the greater fixation of C3b to paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes when whole serum complement is activated, we compared the characteristics of binding of factors B and H to normal and PNH red cells bearing C3b (EC3b). Factor B binding is homogeneous, there is 1:1 stoichiometry, and the affinity constant at equilibrium for factor B binding is the same for normal and PNH EC3b. In contrast, analysis by Scatchard's method of factor H binding results in a curvilinear plot, the deviation from linearity being exaggerated for the PNH EC3b. The heterogeneity of binding of factor H appears to be a consequence of the nonrandom distribution of C3b about the alternative pathway convertase site. This nonrandom distribution does not induce negative cooperativity but rather effects a biophysical milieu which enhances factor H binding. The greater heterogeneity of binding of factor H to PNH E bearing nonrandomly distributed C3b appears to be due to the presence of a greater proportion of lower affinity binding sites on the PNH EC3b. However, it appears unlikely that this greater heterogeneity of factor H binding contributes to the enhanced fixation of C3b to PNH erythrocytes.     

Localization of the heparin-binding site on complement factor H.

Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.     

Differences between the binding sites of the complement regulatory proteins DAF, CR1, and factor H on C3 convertases

Binding studies using purified decay-accelerating factor (DAF), CR1, and Factor H indicate that the primary interaction of DAF with C3 convertases is with the Bb or C2a subunits, whereas CR1 and Factor H interact primarily with the C3b or C4b subunits. The ability of soluble DAF, CR1, or Factor H to decay C3b,Bb bound to zymosan was inhibited by various concentrations of fluid-phase competitors (C3b, Bb, C3b,Bb, C3b,B, C4b, or C4b,C2a) in 0.1% NP-40 at 22 degrees C. The apparent association constants (appKa) for DAF were 0.045, 0.067, 0.91, 0.71, 0.00045, and 0.53 microM-1, respectively. The appKa for CR1 were 0.50, 0.0040, 1, 1, 1, and 1.1 microM-1, respectively. The appKa for Factor H were 4.3, 0.0005, 2.9, 6.3, 0.27, and 0.29 microM-1, respectively. Thus, C3b binds to DAF with a 10-fold lower affinity than to CR1 and a 100-fold lower affinity than to Factor H. The appKa of C3b,Bb for the three proteins were more similar: DAF (0.91 microM-1), CR1 (1 microM-1), and Factor H (2.9 microM-1). DAF binds to Bb with a 50% higher affinity than to C3b, and to C4b,C2a with a 1000-fold higher affinity than to C4b alone. In contrast, CR1 and Factor H bind almost equally well to the C3 convertases and to their noncatalytic subunits. The affinity of DAF for CVF,Bb was similar to its affinity for Bb alone, suggesting that DAF does not recognize conformational determinants unique to Bb in C3 convertases.     

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.     

Species specificity of recognition by the alternative pathway of complement

The recognition function of the alternative complement pathway was studied with isolated human and rabbit components. Zymosan and homologous and heterologous erythrocytes were used as representative activators or nonactivators. The binding affinity of Factor B and Factor H for particle-bound C3b was measured. In both species, the average affinity of Factor H for bound C3b on homologous cells (nonactivators) was eight to 10 times higher than on zymosan particles (activators). The interaction between Factor H and C3b on rabbit erythrocytes was species-specific: rabbit Factor H bound strongly to rabbit C3b on rabbit erythrocytes and also on human erythrocytes, which are nonactivators for the rabbit alternative pathway. Human Factor H bound strongly to human C3b on human erythrocytes but seven times weaker on rabbit erythrocytes, which are activators of the human alternative pathway. No substantial differences were found in the binding of Factor B to bound C3b regardless of the nature of the particle to which C3b was bound. The results indicate that in the two species studied, the molecular mechanism of recognition is analogous and that recognition is species-specific.     

Analysis of recognition in the alternative pathway of complement. Effect of polysaccharide size

Covalent attachment of the complement (C) protein C3b to polysaccharides on biologic particles which activate the alternative pathway leads to changes in the affinity of C3b for factor H, a regulatory protein of the C system. In this study the size of the site with which the polysaccharides interact and its special relationship to the thioester site were investigated using a fluorimetric assay and soluble C3b attached to low m.w. polysaccharides. Oligomers of alpha 1-6 and alpha 1-4 polyglucose and beta 1-2 polyfructose were prepared and attached to C3b at the thioester site. C3b bound to monomeric, dimeric, or trimeric sugars exhibited the same interaction with factor H as free C3b, i.e., there was no effect due to attachment alone. Beginning with tetrameric oligosaccharides a linear decrease in factor H binding was observed with increasing oligosaccharide size and the effect reached an apparent maximum with large polysaccharides. Maximum inhibition of factor H function was estimated to occur at a length of 16 saccharide units. The results suggest that this site, which regulates the inactivation rate of surface-bound C3b and thus the activation of the alternative pathway of C, spans a maximum of 13 sugar units (less than 65 A) starting four units (approximately 15 A) from the thioester site in C3b.     

Factor I co-factor activity of CR1 overcomes the protective effect of IgG on covalently bound C3b residues

We have shown previously that C3b resides in a protected site when it is covalently bound to IgG (C3b-IgG). Such C3b displays a reduced affinity for factor H, with consequent enhanced survival in the presence of factors H and I and increased capacity for promoting alternative pathway consumption of C3. Because erythrocyte CR1 may be a major co-factor for factor I-mediated inactivation of immune complex-borne C3b in blood, we have examined the effect of covalently bound IgG on the C3b-CR1 interaction. Binding of monomeric C3b and C3b-IgG to human erythrocyte CR1 demonstrates identical ionic strength dependence for both species. Identical numbers of binding sites with indistinguishable affinities are detected by both ligands. Cleavage of the alpha'-chain of C3b and the alpha'-heavy chain of C3b-IgG proceeds at the same rate when erythrocyte CR1 serves as co-factor for factor I. Unlike factor H, CR1 supports a second cleavage of fluid-phase iC3b alpha'1 chain (free or bound to IgG) that generates C3c and a 33,000 m.w. fragment, which bears antigenic markers characteristic of C3g. Inactivation of C3b and C3b-IgG by CR1 and factor I also occurs at physiologic ionic strength, but proceeds very slowly relative to rates attainable with sub-physiologic inputs of factor H. CR1 does not recognize IgG-bound C3b as being in a protected site but, because of low binding affinity at physiologic ionic strength, is probably highly dependent on multivalent ligand-receptor interactions to efficiently exert its co-factor functions. Thus, inactivation of C3b-IgG heterodimers or small immune complexes bearing limited numbers of C3b residues may remain largely factor H-dependent in vivo, with resultant enhanced C3b survival.     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.     

The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.

The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.     

CNBr cleavage of the light chain of human complement factor I and alignment of the fragments.

The primary structure of the second component of human complement (C2) was determined by cDNA cloning and sequence analysis. C2 has 39% identity with the functionally analogous protein Factor B. The C-terminal half of C2a is homologous to the catalytic domains of other serine proteinases. C2b contains three direct repeats of approx. 60 amino acid residues. They are homologous to repeats in Factor B, C4b-binding protein and Factor H, suggesting a functional significance of the repeat in C4b and C3b binding. The repeats are also found in the non-complement proteins beta 2-glycoprotein I and interleukin-2 receptor, and this repeat family may be widespread.     

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.     

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.     

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.     

Electron microscopy and hydrodynamic properties of factor XIII subunits

Factor XIII is a transglutaminase important in blood coagulation and fibrinolysis. Its function is to catalyze peptide bond formation between the gamma-carboxamide group of glutamines in one protein and the epsilon-amino group of lysine in another. There are two zymogenic forms of factor XIII: one is a noncovalent, intracellular dimer (A2); the other is a noncovalent, extracellular tetramer (A2B2). The catalytic function resides in the activated A chain (A2.). Purified forms of factor XIII (A2B2, A2, A2.B2, B) were prepared and analyzed by electron microscopy, gel filtration, and gradient centrifugation. Hydrodynamic constants were derived. Electron microscopy of rotary-shadowed molecules showed A2 to consist of two globular particles each about 6 x 9 nm in size. The A2 dimer is significantly elongated, 18 nm long and 6 nm in diameter. Sedimentation and gel filtration of the A2 dimer are consistent with this asymmetric structure. B protein is a filamentous, flexible strand with kinks, with a contour length of 30 nm and a diameter of approximately 2-3 nm. The sedimentation and gel filtration behavior of the B subunit are characteristic of a highly asymmetric molecule. The observed structure of the B subunit, combined with data for its amino acid sequence, suggests a modular structure. The B subunit is a member of a family of proteins composed of tandem, repeating structures (referred to as GP-I domains); the structure seen by electron microscopy for B subunit is probably applicable to all proteins in this family. Plasma and platelet factor XIII zymogens are tetrameric and dimeric, but B protein, in the absence of A protein, appears to be monomeric. Our model for the A2B2 zymogen has the elongated A2 dimer forming the core and the two B strands wrapping around the outside.     

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β₂-glycoprotein I and interferes with innate immune recognition.     

Monoclonal antibodies against the complement control protein Factor H (β1 H)

Two mouse monoclonal antibodies against the human complement control protein, Factor H (beta 1H), are described. The antibodies are both IgG - gamma 1 - subclass and are directed against different epitopes on the human Factor H molecule. One of the antibodies, MRC OX 24, increases the cofactor activity of Factor H in Factor I-mediated cleavage of soluble C3b. The second antibody, MRC OX 23, which has no effect alone, reduces the increase in cofactor activity observed in the presence of the first antibody. However, MRC OX 24 inhibits the binding of 125I-labelled Factor H to surface-bound C3b (EAC3b). Again MRC OX 23 alone does not have an effect but decreases the inhibition in 125I-labelled Factor H binding to EAC3b observed with MRC OX 24. These studies show clearly that the interaction of Factor H with soluble C3b is different to its interaction with surface-bound C3b. In an indirect immunoprecipitation system using these monoclonal antibodies, single-chain molecules of 150 000 mol.wt. are specifically precipitated from human serum and also from the sera of other primates - rhesus monkey, cynomolgus monkey, and African green monkey. There was no precipitation from sera of cow, pig, sheep, chick, or rabbit. Using a radioimmunoassay with radiolabelled monoclonal MRC OX 23, the concentration of Factor H in human plasma was determined.     

Localization of the complement-component-C3b-binding site and the cofactor activity for factor I in the 38kDa tryptic fragment of factor H.

Trypsin treatment of human factor H (H160) [enzyme/substrate ratio 1:100 (w/w), 30 min, 37 degrees C] generated a 38 kDa (H38) and a 142 kDa (H142) fragment linked by disulphide bonds (H38/142). The fragments were purified by reduction with 2-mercapto-ethanol, gel filtration on a Sephadex G-200 column and affinity chromatography with monoclonal anti-(factor H) antibody coupled to Sepharose 4B. This monoclonal antibody bound to a site in the 38 kDa fragment. To localize the C3b binding site in factor H we used two enzyme-linked immunosorbent assays (e.l.i.s.a.). For the first test, e.l.i.s.a. plates were coated with C3b; H160, H38/142, H38 and H142 were added, and their binding was monitored by goat anti-(factor H) and peroxidase-labelled rabbit anti-goat antibodies. Only intact factor H bound to the C3b-coated plates. For the second test, e.l.i.s.a. plates were coated with comparable amounts of factor H or its fragments, and C3b was offered at several dilutions. In contrast with the results from the first assay, C3b bound to intact factor H, H38/142 and H38 but not to H142, thus characterizing H38 as the fragment carrying the C3b-binding site. To identify the fragment responsible for the cofactor activity of factor H (cleavage of fluid-phase C3b by factor I), 125I-C3b was incubated with either H38 or H142 and factor I. H142 had no cofactor activity, whereas H38 had the same cofactor function as intact H. To further investigate the relationship between the C3b-binding site and the site of factor H essential for its cofactor activity, we made use of monoclonal antibodies directed against the H38. Those antibodies inhibiting the binding of C3b to H160 also inhibited the cofactor function, whereas those without effect on the C3b binding also did not interfere with the cofactor activity. This suggests that the C3b-binding site and the site essential for the cofactor activity of factor H are both localized in the 38 kDa tryptic fragment of factor H in close proximity or are identical.     

Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations

Factor H is a regulatory glycoprotein of the complement system. We expressed the three N-terminal complement control protein modules of human factor H (FH1-3) and confirmed FH1-3 to be the minimal unit with cofactor activity for C3b proteolysis by factor I. We reconstructed FH1-3 from NMR-derived structures of FH1-2 and FH2-3 revealing an approximately 105-A-long rod-like arrangement of the modules. In structural comparisons with other C3b-engaging proteins, factor H module 3 most closely resembles factor B module 3, consistent with factor H competing with factor B for binding C3b. Factor H modules 1, 2, and 3 each has a similar backbone structure to first, second, and third modules, respectively, of functional sites in decay accelerating factor and complement receptor type 1; the equivalent intermodular tilt and twist angles are also broadly similar. Resemblance between molecular surfaces is closest for first modules but absent in the case of second modules. Substitution of buried Val-62 with Ile (a factor H single nucleotide polymorphism potentially protective for age-related macular degeneration and dense deposit disease) causes rearrangements within the module 1 core and increases thermal stability but does not disturb the interface with module 2. Replacement of partially exposed (in module 1) Arg-53 by His (an atypical hemolytic uremic syndrome-linked mutation) did not impair structural integrity at 37 degrees C, but this FH1-2 mutant was less stable at higher temperatures; furthermore, chemical shift differences indicated potential for small structural changes at the module 1-2 interface.     

Multifunctional specificity of the protein C/activated protein C Gla domain

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.