Analysis of the mechanism of recognition in the complement alternative pathway using C3b-bound low molecular weight polysaccharides

The human complement (C) system recognizes bacterial, fungal and viral activators of the alternative pathway following covalent attachment of the protein C3b to carbohydrates (CHO) on the surface of the organisms. Recognition first manifests itself as a 3- to 10-fold reduction in the affinity of C3b for factor H, a regulatory protein of C. This report describes the use of a fluorimetric assay which is sensitive to the C3b-H interaction to study the characteristics of recognition. Fluid phase C3b covalently bound to CHO (C3b-CHO) was prepared by activating C3 in the presence of the small homopolymers dextran (alpha 1-6 polyglucose) or inulin (beta 1-2 polyfructose). In particulate form both polysaccharides are activators of C. The conjugates exhibited increased resistance to inactivation in the factor H-dependent assays compared to C3b not bound to CHO and to C3b bound to mono- or disaccharides. The dextran-induced restriction of inactivation was partially reversed by treatment of the conjugate with dextranase. C3b-CHO conjugates failed to bind to factor H-Sepharose and when introduced into serum behaved as though C3b was attached to particulate activators of C, suggesting that the fluorimetric assay accurately reports recognition. The results suggest that the recognition site which induces a reduction in the affinity of C3b for factor H is distinct from the thioester site of C3b and can recognize structural features of polysaccharides including size, sialic acid content, and possibly aspects of three-dimensional oligosaccharide structure.     

Complement factor H-binding protein of Raji cells and tonsil B lymphocytes.

Several reports have indicated that Factor H has specific effects on certain cell populations, suggesting that Factor H receptors may exist. Lambris & Ross [(1982) J. Exp. Med. 155, 1400-1411] purified a protein from Raji B-lymphoblastoid cell culture supernatants, using Factor H-Sepharose affinity chromatography. This species appeared to consist of two disulphide-linked components each of Mr 50,000, with an additional 50,000-Mr chain attached non-covalently. The existence of cell-surface Factor H-binding proteins has now been re-investigated with 125I surface-labelled Raji and tonsil B cells. Non-ionic-detergent extracts of the cells, in 0.1% Nonidet P40/10 mM-sodium phosphate buffer, pH 7.4, were incubated with Factor H-Sepharose in the presence of proteinase inhibitors. After the beads had been washed, bound components were eluted with 50 mM-NaCl. A single radioactive species was eluted from the resin, which migrates identically with Factor H (apparent Mr 170,000) in SDS/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. Biosynthetic radiolabelling studies confirmed that this species was synthesized by Raji cells. Examination of culture supernatants from biosynthetically radiolabelled Raji cells showed again the presence of a single soluble species that bound to Factor H-Sepharose, but this species was of lower Mr (approx. 105,000) than the membrane-derived protein. The soluble form may be produced by proteolysis of the membrane form, or may be of separate origin. The similarity in size of the cell-surface protein to Factor H was initially confusing, but it is distinct from cell-surface Factor H on the basis of three criteria: (1) it is not recognized by anti-(Factor H) monoclonal antibodies MRC OX23 and MRC OX24, nor by polyclonal F(ab')2 anti-(Factor H); (2) it does not bind to Zn2+-chelate resin, whereas Factor H does; (3) cell-surface Factor H present on U937 cells does not bind to Factor H-Sepharose.     

Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b

The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 Å limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.     

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.     

Pig complement regulator factor H: molecular cloning and functional characterization

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1–4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15–20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1–7 and SCR 15–20 proteins, but not the SCR 1–4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.The nucleotide sequence data reported are available in the EMBL database (accession number AJ278470)     

Native C3 does not bind to the C3b receptor (CR1) of human blood B lymphocytes or alter immunoglobulin synthesis

Complement receptors on lymphocytes were first described more than 12 yr ago (1-3) and have come to be used as a common marker for the identification of B cells (4). The function of these receptors on the lymphocyte and their possible role in induction and/or regulation of the immune response remain unclear. In particular, there continues to be controversy as to whether native C3 can bind to the C3b receptor of these cells without cleavage to C3b (5-10). The resolution of this question is critical in order to clarify the expected state of availability of the receptor in vivo, because in plasma, the C3 concentration is relatively high (1.1 to 1.5 mg/ml), whereas there is little or no circulating C3b due to efficient degradation by factor H and the C3-inactivator (11). With the recent development of an improved method for the isolation of C3 from human plasma, it has been possible to obtain biochemically and functionally pure C3 that has not undergone structural or conformational alteration during processing and fully retains the specific hemolytic activity of C3 in fresh serum (12). Berger et al. (13) were able to demonstrate that C3 prepared in this way failed to bind to the C3b receptor of human polymorphonuclear leukocytes or erythrocytes. Similar observations were made by Schreiber et al. (14), also with phagocytic cells and erythrocytes, and by Dixit et al. (15) with an isolated membrane receptor preparation from rabbit macrophages. In the present communication, we extend these observations to human peripheral blood B lymphocytes. Purified C3 in its native state fails to block B lymphocyte-EA (IgM) C4b3b rosettes, whereas C3b causes 50% inhibition at 5 to 6 micrograms/ml. Furthermore, C3 failed to alter polyclonal immunoglobulin (Ig) production by human B cells, whereas C3b inhibited this B cell function. These data suggest that native C3 does not bind to the C3b receptors of B lymphocytes, and thus they are not occupied under normal conditions in vivo.     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1)

We identified on the membrane of mouse spleen cells a polypeptide of Mr 190,000 (S190), with binding affinity for the mouse third component of the complement system (C3). S190, purified by affinity chromatography on C3-Sepharose, has properties resembling those of the human C3 receptor type 1 (CR1). Thus, S190, like CR1, served as a cofactor for the C3b inactivator (I)-mediated cleavage of fluid-phase C3b into iC3b, and had cofactor activity comparable to that of serum factor H (H). S190 also acted as a cofactor for the cleavages of membrane-bound C3b or membrane-bound iC3b into C3c (Mr 140,000) and C3dg (Mr 40,000) by serum factor I. As is the case with CR1, the specific activity of S190 for the cleavages leading to C3c-C3dg formation was approximately 100-fold greater than that of H. We therefore conclude that S190 and CR1 are analogous proteins.     

Targeting of complement to tumor cells by heteroconjugates composed of antibodies and of the complement component C3b

Tumor cells have adapted several strategies which permit them to grow in an immunologically hostile environment. The C system can potentially destroy these cells; however, its action needs to be specifically potentiated on the surface of the tumor cells. To this end, a heteroconjugate composed of a mouse mAb and of the human C3b C component has been generated by using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The two mAb which were used in this study are V1-10 and TIB219 which bind to the human and mouse transferrin receptors, respectively. The mAb-C3b conjugates were purified by gel filtration and were each composed of one mAb and one C3b. They bound to the human K562 and HL60 or mouse ALB1 cell lines and amplified the killing of these cells by C from 10 to 15% to 70 to 100%. Fresh normal human or mouse sera were used as a source of C. The mAb-C3b conjugates activated primarily the alternative pathway of C since only C3 and factor B but not C4 were cleaved in the sera. After disulfide-linking to the mAb, the C3b became highly resistant to inactivation by factors H and I, probably due to its reduced factor H binding capacity. On the other hand, the conjugated C3b bound factor B better than free C3b and produced more C3 convertases which expressed increased stability. These results suggest that mAb-C3b conjugates may serve as an effective tool for the specific activation of the cytolytic C system on selected cells. As such, they may be used in vitro or in vivo to target the autologous C to tumor cells or to lymphocytes and may promote tumor immunotherapy.     

Domain swapping reveals complement control protein modules critical for imparting cofactor and decay-accelerating activities in vaccinia virus complement control protein

Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.     

SARS-CoV-2–encoded ORF8 protein possesses complement inhibitory properties

Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2–encoded ORF8 protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that preincubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway; this preincubation inhibits factor I–mediated proteolysis and blocks factor B zymogen activation into active Bb. ORF8 binding results in the occlusion of both factor H and factor B from C3b, rendering the complexes resistant to factor I–mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum–induced lysis of rabbit erythrocytes with an IC₅₀ value of about 2.3 μM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the β-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the alternative pathway, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.     

Functional characterization of the complement control protein homolog of herpesvirus saimiri: ARG-118 is critical for factor I cofactor activities

Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 microm) as well as to C4b (KD = 0.8 microm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack.     

Regulatory system of guinea-pig complement C3b: two factor I-cofactor proteins on guinea-pig peritoneal granulocytes

Complement factor I is a plasma protease serving for proteolytic inactivation of C3b together with its cofactor. We have identified two factor I-cofactor activities in solubilized extracts of guinea-pig peritoneal granulocytes using guinea-pig factor I (Igp) and fluorescent-labeled methylamine-treated guinea-pig C3 (f-C3(MA)gp). One of these eluted from a chromatofocusing column between pH 7.6-7.1, and the other at about pH 5.7. These two cofactor fractions both interacted with Igp and, to a lesser degree, with human factor I (Ihu) on C3(MA)gp cleaving it into an inactive C3bi analogue, but did not cleave methylamine-treated human C3 (C3(MA)hu) together with Igp or Ihu. These factors are therefore species specific. The neutral and acidic fractions with cofactor activity contained C3(MA)gp-binding proteins with a doublet of 55 kDa and 42 kDa, and a singlet of 160 kDa, respectively, on SDS-PAGE. These proteins may be membrane cofactor protein (MCP) and C3b/C4b receptor (CR1) of guinea-pigs.     

Factor I-mediated processing of complement fragments on HIV immune complexes targets HIV to CR2-expressing B cells and facilitates B cell-mediated transmission of opsonized HIV to T cells

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.     

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.     

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.     

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.     

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.     

Phylogeny of regulatory proteins of the complement system. Isolation and characterization of a C4b/C3b inhibitor and a cofactor from sand bass plasma

We have previously demonstrated that the alpha'-chain of human activated form of the fourth (C4b) and third (C3b) component of C are cleaved by plasma or serum from vertebrate species spanning through 300,000,000 yr of evolution yielding fragments identical with those obtained with human plasma. In this study, we investigated the molecular basis of this reaction. We chose barred sand bass plasma because this is the most primitive species analyzed possessing these activities. Barred sand bass plasma proteins were separated on a Sephadex G-200 column and the eluted samples analyzed for C4b and C3b cleavage. Individual fractions were inactive, but degradation was obtained when proteins of 380 and 155 kDa were combined. In contrast to the human regulatory proteins, the sand bass proteins require Ca2+ ions. K76COOH, an inhibitor of human factor I, inhibited the function of the 155-kDa but not of the 380 kDa-fraction. Thus it appears that the 155-kDa fraction functions as the C4b/C3b cleaving enzyme (I) and the 380-kDa material as its cofactor. Further purification of the 380-kDa fraction yielded a protein that by SDS-PAGE consisted of two noncovalently linked subunits of 110 and 42 kDa at a molecular ratio of 2:1. These two chains were antigenically distinct, and constitute domains of the same protein. The 110-kDa peptide binds C4b and not C3b but it fully expresses the cofactor function for the 155-kDa fraction on the cleavage of both C4b and C3b. Limited tryptic digestion of the 110-kDa domain demonstrated C4b binding activity in fragments of 34, 25, and 23 kDa. The activity of the 34-kDa fragment was the same as that of the undigested protein. Comparison of the amino acid composition of the barred sand bass cofactor and of human C4bp shows similar high content of cysteine and proline but not of tryptophan. It differs from human factor H in cysteine, serine, proline, and tryptophan. These studies indicate that regulatory proteins for the C4b and C3b C fragments may have appeared very early phylogenetically.     

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.     

Ultrastructures and interactions of complement factors H and I.

The human complement regulatory protein, factor H, was examined by high resolution transmission electron microscopy. Results of electron microscopy confirm hydrodynamic analysis and indicate that factor H is a monomer of M(r) approximately 155,000. Factor H is an extended flexible molecule with a contour length of 495 A and a cross-sectional diameter of 34 A. Most images of factor H indicate that its polypeptide chain typically folds back on itself with the result that the average length of a factor H molecule is about half its contour length. Only one end of factor H associates with C3b. When bound to C3b, factor H still shows considerable conformational flexibility. Factor I is a bilobal protein of 130 A in length, and its two globular parts have maximal diameters of 54 and 49 A. The results establish that factor I is a two domain protein where the smaller subunit is a protease and the larger one is involved with binding C3b. Factor I binds C3b with a one-to-one stoichiometry in an ionic strength-dependent fashion. In the absence of sodium chloride an affinity constant of 5.7 x 10(5) M-1 was determined for factor I interaction with C3b. Whereas the Scatchard plot of factor I binding to C3b in the absence of factor H is linear, in the presence of factor H a curvilinear graph is obtained. The strong binding sites on C3b for factor I have an affinity at least 15-fold higher in the presence of factor H than in its absence. The results of both electron microscopy and binding studies were combined to compose a scheme envisioning how factors H and I cooperate for the processing of C3b.