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PubMed1987

Herpes simplex virus glycoproteins gC-1 and gC-2 bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity.

Cells infected with herpes simplex virus type 1 (HSV-1) form rosettes with C3b-coated erythrocytes, whereas cells infected with herpes simplex virus type 2 (HSV-2) or other herpes viruses do not. It was reported that glycoprotein C of HSV-1 (gC-1) mediates the binding of C3b-coated erythrocytes to infected cells and has regulatory (decay-accelerating) activity for the alternative pathway C3 convertase of human complement. We show here that solubilized gC-1 binds to iC3-Sepharose affinity columns. We also report that solubilized gC-2, the genetically related glycoprotein specified by HSV-2, binds to iC3-Sepharose. mAb specific for gC-1 or gC-2 and mutant viral strains were used to identify the C3-binding glycoproteins. In other experiments, HSV-1 mutant strains and recombinants, differing only in their expression of gC, were tested for sensitivity to neutralization by human complement in the presence or absence of antibodies specific for HSV gD. In either case the gC- strain was most sensitive. Expression of gC-1 or gC-2 by isogenic insertion mutants provided protection against complement-mediated neutralization. These results indicate that the genetically and structurally related gC-1 and gC-2 share the functional activity of binding to human C3 and enhance viral infectivity.

Chromatography, Affinity↗
PubMed2002

C3 adsorbed to a polymer surface can form an initiating alternative pathway convertase.

Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.

Adsorption↗
PubMed2004

Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation.

Complement factor B is a 90 kDa protein consisting of three domains: a three-module complement control protein, a von Willebrand factor A domain, and a C-terminal serine protease (SP) domain that adopts a default inactive (zymogen) conformation. The interaction between factor B and pathogen-bound C3b is mediated by its A domain, triggering a conformational change in factor B that ultimately creates the "C3 convertase" of the alternative complement pathway. We report the crystal structure of the A domain from factor B and show that it contains an integrin-like MIDAS motif that adopts the "open" conformation typical of integrin-ligand complexes, with an acidic residue (provided by a fortuitous crystal contact) completing the coordination of the metal ion. Modeling studies indicate that the factor B A domain can also adopt the closed conformation, supporting the hypothesis that an "integrin-like switch" is conserved in complement proteins and perhaps in 60 other A domains found within the human proteome.

Amino Acid Sequence↗
PubMed1988

gp 58/68, a parasite component that contributes to the escape of the trypomastigote form of T. cruzi from damage by the human alternative complement pathway.

A glycoprotein of apparent molecular weight 58,000 (unreduced)/68,000 (in its reduced form) (gp 58/68), which is one of the fibronectin/collagen receptors of Trypanosoma cruzi, was purified to homogeneity from the trypomastigote forms of the Tehuantepec and Y strains of the parasite. Purified gp 58/68 inhibited formation of cell-bound and fluid-phase alternative pathway C3 convertase in a dose-dependent fashion, as assessed using purified human complement components. Gp 58/68 differed from the human regulatory proteins H, DAF, MCP and CR1 and from previously reported regulatory proteins on the parasite membrane in that it was unable to enhance decay-dissociation of preformed alternative pathway C3 convertase sites, did not serve as a co-factor for I-mediated cleavage of C3b and had no inhibitory activity on the classical pathway convertases. The inhibitory effect of gp 58/68 was most likely dependent on an interaction of the protein with factor B rather than with C3b. Gp 58/68 provides trypomastigotes with an additional potential mechanism for escaping complement lysis by the human alternative pathway.

Animals↗
PubMed1988

Developmentally regulated expression by Trypanosoma cruzi of molecules that accelerate the decay of complement C3 convertases.

We recently showed that culture-derived metacyclic trypomastigotes (CMT), but not epimastigotes (Epi), of the Miranda 88 strain of Trypanosoma cruzi evade lysis by the human alternative complement pathway because of inefficient binding of factor B to complement component C3b on the parasite surface. These results suggested that CMT and tissue-culture-derived trypomastigotes (TCT), which also activate the alternative pathway poorly, might produce a molecule capable of interfering with factor B binding to C3b. We now demonstrate that CMT and TCT lysates, as well as molecules spontaneously shed from CMT and TCT but not Epi, accelerate decay of 125I-labeled factor Bb from the alternative-pathway C3 convertase (C3bBb) assembled on zymosan or Epi and also accelerate decay of the classical-pathway C3 convertase (C4b2a) on sheep erythrocytes. Parasites metabolically labeled with [35S]methionine spontaneously shed a limited number of radioactive components ranging in molecular mass from 86 to 155 kDa for trypomastigotes and 25 to 80 kDa for Epi. Decay-accelerating activity within supernatants is inactivated by papain and is coeluted with 35S-containing polypeptides on FPLC anion-exchange chromatography, suggesting that the active constituents are protein molecules. Molecules with decay-accelerating activity may explain the developmentally regulated resistance to complement-mediated lysis in infective and vertebrate stages of the T. cruzi life cycle.

Animals↗
PubMed1986

In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

Animals↗
PubMed1992

Analysis of the effects of activation of the alternative pathway of complement on erythrocytes with an isolated deficiency of decay accelerating factor.

E from individuals with the Inab blood group phenotype have an isolated deficiency of decay accelerating factor (DAF, CD55). DAF is a glycosyl phosphatidylinositol anchored membrane protein that inhibits activation of both the classical and alternative pathways of complement. Deficiency of DAF from the E of paroxysmal nocturnal hemoglobinuria (PNH) is thought to contribute to their greater sensitivity to complement-mediated lysis. Unlike PNH E, however, Inab cells are not susceptible to acidified serum lysis, a process that is mediated through activation of the alternative pathway. This observation led us to hypothesize that membrane constituents other than DAF control susceptibility to acidified serum lysis. To investigate this hypothesis, Inab E were incubated in acidified serum, and hemolysis and C3 deposition (as a measure of alternative pathway activation) were quantitated. C3 deposition of Inab cells was approximately 20 times greater than normal, however, hemolysis was not observed. Inab E expressed a normal amount of membrane inhibitor of reactive lysis (MIRL, CD59), a glycosyl phosphatidylinositol anchored protein that is also deficient in PNH. When MIRL function was blocked with antibody, C3 deposition markedly increased, and 100% of the Inab cells hemolyzed in acidified serum. These studies demonstrate that susceptibility to acidified serum lysis is controlled primarily by MIRL, and that in addition to its regulatory affect on the membrane attack complex, MIRL also modulates the activity of the C3 convertase of the alternative pathway by a mechanism that remains to be determined.

Animals↗
PubMed1977

Pathways of complement activation in membranoproliferative glomerulonephritis and allograft rejection.

The complement system is comprised of at least 18 plasma proteins and consists of four functional divisions: two pathways for activation (classical and alternative), a common amplification mechanism for the activating pathways, and a final common effector pathway to which the activating and amplifying sequences are directed. The classical pathway is activated by certain antigen-antibody complexes, while the alternative pathway may be initiated non-immunologically by various microbial polysaccharides. Indeed, mixtures of purified C3, B, D, and P regulated to low-grade interaction by the presence of C3bINA and beta1 H respond to zymosan with amplified C3 and B inactivation. Both pathways form enzymes termed C3 convertases that cleave C3 to generate its major fragment, C3b. C3b interacts with each C3 convertase to permit C5 cleavage in activation of the effector complement sequence, and it interacts with alternative-pathway factors B and D to generate additional C3 convertase, C3bBb, in the amplification pathway. As C3 cleavage represents the most critical step in the elaboration of the biologic effects of the complement system, modulation of this reaction by generation, stabilization, and inactivation of the amplification convertase C3bBb may well determine whether initial activation of the complement sequence eventuates in beneficial or detrimental effects for the host. Initial generation of C3bBb is dependent on prior cleavage of C3, which may occur by the classical pathway or the alternative pathway. Stabilization of C3bBb is achieved with either P or C3NeF after their binding to C3b and C3bBb, respectively. Control of this amplifying step occurs at three levels: intrinsic decay of the inherently labile C3bBb complex, extrinsic decay-dissociation of Bb from the complex by beta1H, and inactivation of C3b by C3bINA. In the presence of stabilizing factors the control proteins must function in sequence, since C3bINA cannot act on C3bBb; beta1H-mediated decay of protective Bb must precede C3b inactivation by C3bINA. C3NeF, which is found in the sera of some patients with MPGN and persistent depressions of serum C3, circumvents all three controls because of its capacity to create a stabilized convertase that is relatively resistant to decay-dissociation by beta1H. The effector complement sequence is activated by cleavage of C3 and C5, which releases vasoactive and chemotactic peptides, C3a and C5a, and generates the major fragments C3b and C5b. C3b, in addition to its function in the amplifying reaction and the C5 convertases, mediates immune adherence to cells possessing membrane-associated receptors for C3b; this in turn promotes the phagocytic and secretory functions unique to each cell type. Cell-bound C5b serves to assemble the cytolytic complex C5b6789, while fluid-phase C5d generates the hemolytically inactive chemotactic complex C567d...

Complement C3↗
PubMedDate not supplied

[Determination of proteinase activity of complement system by immunoenzyme methods].

Modern ELISA for determination of functional activity of component C2 and factors B and D, proteinases of a complement system, and component C3, substrate C3-convertases, key complex enzymes of the complement, have been developed. Essential feature of C3-convertases classical (C4bC2a) and alternative (C3bBb) pathways of the complement activation is that their substrate C3 after proteolytic cleavage is converted into C3b, carrying on the surface thioester covalent bond linking C3b with nucleophilic acceptors that results in immobilization of this proteolytic product near the activating enzyme. Cascade character of an activation of complement system allows to create artificial deficit of separate components in the experimental system and to determine (by ELISA) covalently immobilized component C3 during activation, and also to determine functional activity of any of pre-exhausted components. Use of such approach resulted in the development of the ELISA systems suitable for determination of functional activity of component C2 of classical pathway and factors B and D of the alternative pathway by testing quantity of the immobilized C3b at excess C3. The developed methods allow to investigate mechanisms of functioning of complement, inhibition of the cascade activation by endogenic and exogenous inhibitors, and also to find functional deficiency of components in serum and other biological fluids.

Animals↗
PubMed1992

Modulation of complement activation on hemodialysis membranes by immobilized heparin.

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

Biocompatible Materials↗
PubMed1992

Expression and localization of proteins of the complement system in human skin.

The complement system participates in the immune recognition of foreign antigens, many of which may penetrate the skin by physical injury or transcutaneous adsorption. In this study, we examined the presence of complement components and complement regulatory proteins in the human skin and cultured human keratinocytes. Immunofluorescence studies showed C3, Factor B, decay accelerating factor, the C3b receptor (CR1), and C3d receptor (CR2), distributed among cells of the epidermis as well as on cultured keratinocytes. Immunoblot analysis of keratinocytes supernatants showed the presence of C3 with a molecular weight of approximately 180 kD. The decay accelerating factor was localized as previously reported on elastic fibers; additionally it was observed in the basement membrane zone. In situ hybridization studies suggest the expression of CR1 and CR2 mRNA in human epidermis. These results show the presence in the human epidermis of complement components that are capable of generating the initial C3 convertase of the alternative pathway. The presence of complement regulatory proteins could endow keratinocytes with immune functions such as the regulation of complement activation and endocytosis of C3 opsonized particles.

Antigens, CD↗
PubMed1984

Complement activation by heat-killed human kidney cells: formation, activity, and stabilization of cell-bound C3 convertases.

Heat-killed kidney cells (HKKC) incubated with serum or purified C3 and factors B and D stain intensely for C3 by immunofluorescence. Binding of complement (C) did not occur if ethylenediaminetetraacetic acid (EDTA) was added to the C source or if factor B or D was omitted from the mixture of alternative pathway components. When the C-treated cells were washed, then incubated in purified C3 containing 10 mM EDTA, C3 conversion ensued, confirming the presence of a cell-bound C3 convertase. C3-reactive cells were not generated if EDTA was present during formation of the convertase. The addition of C3 nephritic factor (C3NeF) during formation of the C3 convertase from alternative pathway components greatly enhanced subsequent C3 cleavage, presumably by stabilizing labile C3bBb sites; however, binding of C3NeF did not appear to extend the half-life of the convertase, which was 35 min at 37 degrees C in each case. In contrast, little or no effect on C3 conversion could be detected after the addition of equivalent C3NeF to serum and HKKC. Thus, C activation by HKKC involves formation and activity of a cell-bound C3 convertase, which under certain circumstances can be stabilized by C3NeF.

Cells, Cultured↗
PubMed1989

Evasion of Trypanosoma cruzi from complement lysis.

This review outlines: a) the main biochemical and biological properties of the complement system (C) components; b) the manner through which they interact in the two distinct routes of C activation, the classical and the alternative pathways, to generate the enzymes C3 and C5 convertases responsible for release of the peptides C4a, C3a and C5a endowed with the properties of mediating the early events of the inflammatory process or the potentially cytolytic complex C5b-C9; c) the main features of control of these activation processes; d) the identification of cell surface components present in the trypomastigote forms of Trypanosoma cruzi possibly involved in the mechanisms developed by this parasite to evade C lysis; e) the inactivation or removal of these cell surface components by enzymatic (trypsin or papain), chemical (periodate) or physical (heating at 45 degrees C) treatments; f) isolation of these components by chromatographic methods; and, g) demonstration that some of these cell surface components interfere with C3 convertase formation or action in a manner similar to the decay accelerating factor (DAF).

Animals↗
PubMed1984

Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.

We have examined the role of macrophage (M phi plasma membrane receptors for the cleaved third complement component (iC3b; CR3) and mannosyl, fucosyl terminated glycoproteins (MFR) in uptake of unopsonized zymosan. Monoclonal antibodies against CR3, M1/70 (Mac-1) and MO1, each inhibited approximately 50% of uptake of 125I-zymosan by murine and human M phi, respectively. Yeast mannan inhibited 0-50% of zymosan uptake in various M phi, in parallel with their expression of MFR. We demonstrated that M phi were the source of C3 in our assay and that the activity of other components of the complement system, namely a C3 convertase, factor I, and a factor I cofactor were also present in serum-free cultures of human monocytes. Macrophage C3 was deposited rapidly, within 10 min, on the zymosan particles and mediated binding, ingestion, and stimulation of superoxide release in BCG-activated and thioglycollate-elicited peritoneal M phi via CR3. Local secretion of complement proteins by M phi themselves can therefore opsonize pathogens and cells able to activate the alternative pathway and effect their destruction.

Animals↗
PubMed1986

The effects of metal ions and temperature on the interaction of cobra venom factor and human complement factor B.

An alternative pathway C3 convertase is formed by the equilibrium association of Factor B with cobra venom factor (CVF) followed by the activation step catalyzed by Factor D. However, the association of Factor B with CVF has only occasionally been demonstrated and has not been quantitatively analyzed. Here we show that in the absence of metals the two proteins have significant affinity for each other and reversibly associate in a one-to-one stoichiometry with a dissociation constant of 11.6 microM. Upon the addition of metal ions, the complex is stabilized only 2- to 30-fold in the order Ni2+(Kd = 6.6 microM) less than Mg2+(Kd = 1.1 microM) less than Mn2+(Kd = 0.4 microM). These results suggest that metal ions may be less important in stabilizing the CVF.B complex and more important in promoting the subsequent equilibrium association of CVF.B with Factor D. The stability of the CVF.B complex is variously dependent on temperature in the range studied (14-21 degrees C) depending on the metal ion that is present. The complex formation was demonstrated in the analytical ultracentrifuge at sedimentation equilibrium employing a combination of single- and multiple-independent variable nonlinear least squares analytical techniques. Two different numerical approaches gave very similar results.

Complement Factor B↗
PubMed1986

A reexamination of the role of magnesium in the human alternative pathway of complement.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

Centrifugation, Density Gradient↗