The catalytically active serine protease domain of human complement factor I

Factor I (fI) is a major regulator of complement. As a protease it has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH). Cleavage of C3b by fI yields iC3b, a major opsonin. The cleavage occurs through the formation of a ternary complex between the enzyme, the substrate, and the cofactor. The catalytic subunit of fI, the SP domain, accommodates substrate recognition and cleavage. The role of the fI heavy chain within the catalysis complex is unknown. Using partial proteolysis and affinity chromatography an intact form of the SP domain was generated and isolated from fI in high yield. fI and the SP domain were found to have similar amidolytic activities but strikingly different proteolytic activities on C3(NH(3)). fI did not cleave C3(NH(3)) in the absence of fH, while in its presence it cleaved C3(NH(3)) rapidly at two sites. The SP domain, however, slowly cleaved C3(NH(3)) in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. Pefabloc SC and antipain inhibited the proteolytic activity of both fI and the SP domain, but suramin inhibited only fI and not the SP domain. The contrast in the proteolytic activities suggests that the heavy chain domains and the cofactor must have roles in orienting the natural substrates and restricting cleavage to the two sites which yield iC3b through a highly specific catalysis.     

Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic

Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.     

Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.     

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.     

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.     

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis

Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.     

Regulation of proteolytic activity of complement factor I by pH: C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) have different pH optima for factor I-mediated cleavage of C3b

C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) are integral membrane glycoproteins with factor I-dependent cofactor activity. They bind to C3b, allowing factor I to cleave C3b at two sites (first and second cleavage), which results in the generation of C3bi, a hemolytically inactive form which is a ligand for complement receptor type three (CR3). C3bi is further degraded by factor I and CR1 (third cleavage) to C3dg (a ligand for complement receptor type two, CR2) and C3c. Using two different substrates, fluid-phase C3b and cell-bound C3b, the cleavage of C3b by MCP and factor I was compared to that by CR1 and factor I under various conditions. The optimal pH for the first and second cleavage of either substrate was 6.0 for MCP and 7.5 for CR1. The third cleavage was mediated only by CR1 and factor I, the optimal pH being 8.0. Low ionic conditions enhanced the C3b binding and cofactor activity of both CR1 and MCP. The efficiency of binding C3b to CR1 or MCP was maximal at pH 6.2. The isoelectric point (pI) of MCP was acidic (approximately 4.0), while that of CR1 was 6.8. Therefore, compared to CR1, MCP possesses distinct functional profiles relative to C3b-binding and factor I-cofactor activity.     

Enzymic assay of C3b receptor on intact cells and solubilized cells.

The C3b receptor of human erythrocytes is known to act as a cofactor for the cleavage of the complement protein C3b by the serine proteinase C3b/C4b Ina. The same cofactor activity is shown to be present on human tonsil B-lymphocytes. The cofactor activity of the C3b receptor can be assayed, on intact cells or in solubilized extracts of cells, by determining the rate of C3b cleavage in the presence of fixed concentrations of C3b and of C3b/C4b Ina. This assay method was used to compare the characteristics and relative quantities of C3b receptors on erythrocytes and lymphocytes. The cofactor activities associated with these two cell types resemble each other, but are distinct from the serum cofactor proteins, C4bp and Factor H, in antigenicity and in pH- and ionic-strength-dependence, and are distinct from Factor H in substrate specificity. Assay of cofactor activity in intact cells indicates that there are about 80-fold more receptors per cell on the lymphocyte surface than on erythrocytes. Assays with cells made permeable by detergent show that, whereas essentially all of the receptors on erythrocytes are on the cell surface, B-lymphocytes contain a large internal receptor pool, which makes up more than 80% of the total cofactor activity of the cell.     

Functional properties of membrane cofactor protein of complement.

Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.     

Each of the three binding sites on complement factor H interacts with a distinct site on C3b

Factor H (fH) restricts activation of the alternative pathway of complement at the level of C3, both in the fluid phase and on self-structures, but allows the activation to proceed on foreign structures. To study the interactions between fH and C3b we used surface plasmon resonance analysis (Biacore(R)) and eight recombinantly expressed fH constructs containing fragments of the 20 short consensus repeat domains (SCRs) of fH. We analyzed the binding of these constructs to C3b and its cleavage products C3c and C3d. Three binding sites for C3b were found on fH. Site 1 was localized to the five amino-terminal SCRs (SCR1-5), and its reciprocal binding site on C3b was found to be lost upon the cleavage of C3b to C3c and C3d. Site 2 on fH was localized by exclusion probably within or near SCRs 12-14 (fragment SCR8-20 bound to C3b, C3c, and C3d; SCR8-11 did not bind to C3b at all; and SCR15-20 bound only to the C3d part of C3b). Site 3 on fH for C3b was localized to the carboxyl-terminal SCRs 19-20, and its reciprocal binding site was mapped to the C3d part of C3b. In conclusion, we confirmed and mapped three binding sites on fH for C3b and demonstrated that the three binding sites on fH interact with distinct sites on C3b. Multiple reciprocal interactions between C3b and fH can provide a basis for the different reactivity of the alternative pathway with different target structures.     

Mutations in alpha-chain of C4BP that selectively affect its factor I cofactor function

C4b-binding protein (C4BP) inhibits all pathways of complement activation, acting as a cofactor to the serine protease factor I (FI) in the degradation of activated complement factors C4b and C3b. C4BP is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain, the alpha- and beta-chains being composed of eight and three complement control protein (CCP) domains, respectively. In previous studies we have localized cofactor activity and binding of C4b to alpha-chain CCP1-3 of C4BP, whereas the binding of C3b required additionally CCP4. Likewise, introduced point mutations that decreased binding of C4b/C3b caused a decrease in cofactor activity. In the present study, we describe two mutants of C4BP, K126Q/K128Q and F144S/F149S, clustered on alpha-chain CCP3, which selectively lost their ability to act as cofactors in the cleavage of both C4b and C3b. Both mutants show the same binding affinity for C4b/C3b as measured by surface plasmon resonance and have the same inhibitory effect on formation and decay of the classical pathway C3-convertase as the wild type C4BP. It appears that C4b and C3b do not undergo the same conformational changes upon binding to the C4BP mutants as during the interaction with the wild type C4BP, which then results in the observed loss of the cofactor activity.     

Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46).

Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human complement factor I glycosylation: structural and functional characterisation of the N-linked oligosaccharides

Factor I (fI) is a key serine protease that modulates the complement cascade by regulating the levels of C3 convertases. Human fI circulates in plasma as a heavily N-glycosylated (25-27% w/w) heterodimer composed of two disulphide linked chains, each carrying three N-linked oligosaccharide chains. It had been suggested that the oligosaccharides may have both structural and functional roles in the interactions with the natural substrate and the cofactor during a catalysis. The N-linked glycans of each fI chain were characterised in detail and the analysis revealed a similar composition of the glycan pools with both chains heavily sialylated. Disialylated structures were in excess over monosialylated ones: 55% over 40% for the heavy chain and 62% over 35% for the light chain. The dominant type of glycan identified on both chains was A(2)G(2)S(2), a biantennary structure with chains terminating in sialic acid linked to galactose. The glycan characterisation facilitated a strategy for the partial deglycosylation of the enzyme. Assessment of the proteolytic activities of the native and partially deglycosylated forms of fI showed that both forms of the enzyme have very similar proteolytic activities against C3(NH(3)) indicating that the charged glycans of fI do not influence the fI-cofactor-substrate interactions.     

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.     

Dissection of Functional Sites in Herpesvirus Saimiri Complement Control Protein Homolog

Herpesvirus saimiri is known to encode a homolog of human complement regulators named complement control protein homolog (CCPH). We have previously reported that this virally encoded inhibitor effectively inactivates complement by supporting factor I-mediated inactivation of complement proteins C3b and C4b (termed cofactor activity), as well as by accelerating the irreversible decay of the classical/lectin and alternative pathway C3 convertases (termed decay-accelerating activity). To fine map its functional sites, in the present study, we have generated a homology model of CCPH and performed substitution mutagenesis of its conserved residues. Functional analyses of 24 substitution mutants of CCPH indicated that (i) amino acids R118 and F144 play a critical role in imparting C3b and C4b cofactor activities, (ii) amino acids R35, K142, and K191 are required for efficient decay of the C3 convertases, (iii) positively charged amino acids of the linker regions, which are dubbed to be critical for functioning in other complement regulators, are not crucial for its function, and (iv) S100K and G110D mutations substantially enhance its decay-accelerating activities without affecting the cofactor activities. Overall, our data point out that ionic interactions form a major component of the binding interface between CCPH and its interacting partners.     

Residue Met(156) contributes to the labile enzyme conformation of coagulation factor VIIa

Serine protease activation is typically controlled by proteolytic cleavage of the scissile bond, resulting in spontaneous formation of the activating Ile(16)-Asp(194) salt bridge. The initiating coagulation protease factor VIIa (VIIa) differs by remaining in a zymogen-like conformation that confers the control of catalytic activity to the obligatory cofactor and receptor tissue factor (TF). This study demonstrates that the unusual hydrophobic Met(156) residue contributes to the propensity of the VIIa protease domain to remain in a zymogen-like conformation. Mutation of Met(156) to Gln, which is found in the same position of the highly homologous factor IX, had no influence on the amidolytic and proteolytic activity of TF-bound VIIa. Furthermore, the mutation did not appreciably stabilize the labile Ile(16)-Asp(194) salt bridge in the absence of cofactor. VIIa(Gln156) had increased affinity for TF, consistent with a long range conformational effect that stabilized the cofactor binding site in the VIIa protease domain. Notably, in the absence of cofactor, amidolytic and proteolytic function of VIIa(Gln156) were enhanced 3- and 9-fold, respectively, compared with wild-type VIIa. The mutation thus selectively influenced the catalytic activity of free VIIa, identifying the Met(156) residue position as a determinant for the zymogen-like properties of free VIIa.     

Plasminogen is a complement inhibitor

Plasminogen is a 92-kDa single chain glycoprotein that circulates in plasma as a zymogen and when converted to proteolytically active plasmin dissolves preformed fibrin clots and extracellular matrix components. Here, we characterize the role of plasmin(ogen) in the complement cascade. Plasminogen binds the central complement protein C3, the C3 cleavage products C3b and C3d, and C5. Plasminogen binds to C3, C3b, C3d, and C5 via lysine residues, and the interaction is ionic strength-dependent. Plasminogen and Factor H bind C3b; however, the two proteins bind to different sites and do not compete for binding. Plasminogen affects complement action in multiple ways. Plasminogen enhanced Factor I-mediated C3b degradation in the presence of the cofactor Factor H. Plasminogen when activated to plasmin inhibited complement as demonstrated by hemolytic assays using either rabbit or sheep erythrocytes. Similarly, plasmin either in the fluid phase or attached to surfaces inhibited complement that was activated via the alternative and classical pathways and cleaved C3b to fragments of 68, 40, 30, and 17 kDa. The C3b fragments generated by plasmin differ in size from those generated by the complement protease Factor I, suggesting that plasmin-mediated C3b cleavage fragments lack effector function. Plasmin also cleaved C5 to products of 65, 50, 30, and 25 kDa. Thus, plasmin(ogen) regulates both complement and coagulation, the two central cascade systems of a vertebrate organism. This complement-inhibitory activity of plasmin provides a new explanation why pathogenic microbes utilize plasmin(ogen) for immune evasion and tissue penetration.     

Functional significance of factor H binding to Neisseria meningitidis

Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity.     

Phylogeny of C4b-C3b cleaving activity: similar fragmentation patterns of human C4b and C3b produced by lower animals

Functional and structural studies of the activated proteins of the complement system C4b and C3b have led to the identification of cleavage products resulting from the effect of the regulatory proteins, factor I, H, and C4b binding protein (bp). In this paper we report the results of studies that investigated the capacity of plasma or serum from a wide range of phylogenetic species to yield similar cleavage products. Sera and plasma from mammals, reptiles, amphibia, and fishes are capable of cleaving fluid phase human C4b and C3b, generating apparently the same fragments as observed using normal human serum: alpha 2, alpha 3, alpha 4 from the alpha' chain of C4b: and alpha-68, alpha-46, alpha-43, and alpha-30 from the alpha' chain of C3b. When C3b bound to a cell membrane is used C3c and C3dg are generated. The generation of these fragments from C3bi is a dose-dependent reaction. There is no correlation between the evolution of the species and the quantitative capability to degrade the substrates. Birds possess only a limited capability to degrade the alpha' chain of C4b and have no cleaving activity for C3b, whereas sera from more primitive vertebrate species (chondrichthyes and agnatha) fail to participate in the reaction. Contrary to other species, the proteins in fish serum or plasma responsible for the degradation of C4b and C3b show a unique requirement for Ca2+ ions. Magnesium and barium are less effective, and in their presence a 65,000 dalton intermediate product is observed. These results demonstrate that protein(s) displaying proteolytic activity for products of complement activation, probably related to I, H, and C4bp, are present in plasma of species whose evolution have preceded humans by 300 million years. Moreover, the recognition of human substrates and the generation of fragments identical to those produced by human serum suggests that human C4b and C3b share structural characteristics with their evolutionary ancestors in the serum or plasma of the species studied.     

Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex

Structural and mutagenesis data have indicated that the 220-loop of thrombin is stabilized by a salt-bridge between Glu-217 and Lys-224, thereby facilitating the octahedral coordination of Na (+) with contributions from two carbonyl O atoms of Arg-221a and Lys-224. All three residues are also conserved in fXa and the X-ray crystal structure of fXa indicates that both Glu-217 and Lys-224 are within hydrogen-bonding distance from one another. To investigate the role of these three residues in the catalytic function of fXa and their contribution to interaction with Na (+), we substituted them with Ala and characterized their properties in both amidolytic and proteolytic activity assays. The results indicate that the affinity of all three mutants for interaction with Na (+) has been impaired. The mutant with the greatest loss of affinity for Na (+) (E217A or E217Q) also exhibited a dramatic impairment ( approximately 3-4 orders of magnitude) in its activity toward both synthetic and natural substrates. Interestingly, factor Va (fVa) restored most of the catalytic defect with prothrombin, but not with the synthetic substrate. Both Glu-217 mutants exhibited a near normal affinity for fVa in the prothrombinase assay, but a markedly lower affinity for the cofactor in a direct-binding assay. These results suggest that, similar to thrombin, an ionic interaction between Glu-217 and Lys-224 stabilizes the 220-loop of fXa for binding Na (+). They further support the hypothesis that the Na (+) and fVa-binding sites of fXa are energetically linked and that a cofactor function for fVa in the prothrombinase complex involves inducing a conformational change in the 220-loop of fXa that appears to stabilize this loop in the Na (+)-bound active conformation.