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PubMed1985

The cobra complement system: I. The alternative pathway of activation.

The complement system of the cobra snake is of particular interest because cobra venom contains cobra venom factor (CVF), a protein that is related to C3 and forms a stable C3 convertase with mammalian Factor B. We investigated the alternative pathway of cobra complement. Cobra plasma lysed erythrocytes in presence of Mg-EGTA of some mammalian species, but not cobra erythrocytes. The hemolytic activity was inhibited by EDTA and destroyed by heating. Preincubation of cobra plasma with alternative pathway activators (zymosan, inulin, lipopolysaccharide), with small nucleophiles (methylamine, hydrazine), or with chaotropes (KSCN) abrogated the hemolytic activity. The activity of cobra plasma was not affected by CVF. Cobra plasma also lysed EAC1423 cells in presence of EDTA but not EAC142 cells (prepared with sheep erythrocytes, rabbit antibody, and human complement proteins) indicating the presence of C5 in cobra plasma that is susceptible to activation by the human C5 convertase. These results indicate that the cobra has a complement system with an alternative pathway very similar to mammalian complement.

Animals↗
PubMed1994

The interaction of glycoprotein C of herpes simplex virus types 1 and 2 with the alternative complement pathway.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) binds the human complement fragment C3b, but the two proteins differ in their ability to bind C3b on infected cell surfaces. In addition, gC-1, but not gC-2, accelerates the decay of the alternative pathway C3 convertase, thereby affecting later steps of the complement cascade. Previously, we constructed linker insertion and deletion mutants of gC-1 and gC-2 and used transient transfection to express mutant proteins in uninfected cells. In spite of the differences between gC-1 and gC-2, C3b binding was localized to residues within the central portion of both proteins, encompassing the first four cysteines. For gC-1, deletion mutants lacking amino acids 33 to 123 or 367 to 469 or lacking both regions still bound C3b. We recombined these deleted forms of gC-1 into gC-39, an HSV-1 strain lacking the gC gene. The altered forms of gC-1 were incorporated into virions, expressed on the surface of infected cells, and bound C3b. We used these proteins to investigate the structural basis for the inhibitory action of gC-1 on the complement cascade. We found that gC-1 does not inhibit formation of the alternative pathway C3 convertase. This convertase is stabilized by the serum protein properdin. Purified gC-1, but not gC-2, inhibits the binding of properdin to C3b, suggesting that this destabilizes the convertase. The mutant lacking amino acids 367 to 449 was able to inhibit properdin binding to a limited extent when present at high concentrations, although it bound to C3b more weakly than wild-type gC. In contrast, the protein lacking amino acids 33 to 123 was unable to inhibit properdin binding to C3b. Thus, gC-1 contains two structural domains, one for C3b binding, residues 124 to 366, and another, residues 33 to 133, which interferes with properdin binding to C3b.

Animals↗
PubMed1985

Inhibition of C3 deposition on solid-phase bound immune complexes by lactoferrin.

In the study described here, the effect of human tears and purified lactoferrin was investigated on the deposition of complement components on solid-phase bound immune complexes. After incubating immune complexes with fresh normal human serum, the deposition of complement components (C3, C4 and C5) was measured with an ELISA technique. Rabbit antibodies were used as a constituent of the immune complexes, and so both alternative and classical complement pathway activation could be studied. The addition of human tears or purified lactoferrin to this system resulted in the inhibition of classical pathway deposition of C3 and C5, while C4 deposition was not affected. High concentrations of human tears also inhibited alternative pathway C3 deposition on immune complexes, whereas lactoferrin did not detectably affect this pathway. The inhibition of complement activation by tears was not due to a masking of the immune complexes or the already deposited C3. Experiments with purified lactoferrin furthermore showed that lactoferrin did not bind to the complexes, either before or during complement activation. These findings suggest that the complement inhibitory effect is probably taking place in the fluid phase. Saturation of lactoferrin with iron or copper ions resulted in a markedly diminished effect on the capacity of lactoferrin to inhibit complement activation. C4 deposition on immune complexes was not affected by lactoferrin, which suggested that the inhibition of the classical pathway was due to an effect on the classical C3 convertase. The fact that lactoferrin inhibits the classical, but not the alternative C3 convertase, suggests that the effect is probably not mediated through a competition for certain trace metal ions, but may be caused by protein-protein interactions. The findings reported here indicate that lactoferrin may play an important anti-inflammatory role by modulating activation of the complement system. This observation adds a new property to the already described functions of the iron-binding protein lactoferrin.

Antigen-Antibody Complex↗
PubMed1986

Production of nephritic factor of the alternative complement pathway by Epstein Barr virus-transformed B cell lines derived from a patient with membranoproliferative glomerulonephritis.

Nephritic factor of the alternative complement pathway (C3NeF) is an IgG autoantibody which binds to and stabilizes the C3 convertase (C3bBb) enzyme, and which has been detected mainly in sera from patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy. To study the production of C3NeF, mononuclear cells isolated from the peripheral blood of patients with MPGN and C3NeF activity in their sera were infected with Epstein Barr virus (EBV) to establish active B lymphocyte cell lines. By using a modified C3NeF screening assay, we detected C3NeF activity in the supernatant of a B cell line derived from a patient with MPGN Type II, but in none of the supernatants of B cell lines derived from normal individuals. C3NeF-positive supernatants were investigated for their ability to conserve classical or alternative pathway C3 convertase activity by using EAC3bBb and EAC4b2a stabilization assays. C3NeF-positive supernatants stabilized the C3bBb convertase activity, but not the C4b2a convertase activity. Studies of the supernatants, using anti-human IgG affinity columns, showed that the C3NeF activity was in the IgG fraction; furthermore, C3NeF antibody agglutinated sheep erythrocytes coated with C3bBb, but not with C3b alone. On gel electrophoresis, both heavy and light chains of the C3NeF were comparable in size to that of normal human IgG molecules. We conclude that C3NeF, produced in vitro by EBV-transformed B cell lines derived from a patient with MPGN Type II, is functionally identical to the conventional C3NeF in serum. In vitro preparation of homogeneous NeF(s) should greatly facilitate the studies of the role of these autoantibodies in complement dysmetabolism.

B-Lymphocytes↗
PubMed1991

Comparative study of in vitro inhibition of activation of the classical and alternative pathways of human complement by the magnesium and sodium salts of the anti-inflammatory peptide N-acetyl-aspartyl-glutamic acid (NAAGA).

The inhibitory activity of the sodium salt of the anti-inflammatory peptide N-acetyl-aspartyl-glutamic acid (NAAGA) on activation of the classical and alternative pathways of human complement was compared with that of the clinically used magnesium salt of NAAGA (NAAGA-Mg). Sodium salt of NAAGA (NAAGA-Na) inhibited both pathways of activation in a dose-dependent manner at concentration ranging from 1 to 10 mM by acting on formation and/or function of the C3 convertases as shown by the inhibitory capacity of the peptide on the release of the C3 cleavage fragment C3b and C3a. NAAGA-Na was as effective as NAAGA-Mg in inhibiting classical pathway activation at concentration above 10 mM. NAAGA-Na was more effective than NAAGA-Mg in inhibiting the alternative pathway since the sodium salt did not interfere with Mg-dependent formation of the alternative pathway C3 convertase.

Animals↗
PubMed1978

Complement C3 convertase: cell surface restriction of beta1H control and generation of restriction on neuraminidase-treated cells.

The alternative or properdin pathway of complement is primarily controlled by the endopeptidase C3b inactivator (C3bINA) and the nonproteolytic glycoprotein beta1H. The molecular mechanisms of control were investigated by performing binding studies of radiolabeled complement proteins to C3b bearing sheep erythrocytes (E(S)C3b). C3b was found to have distinct binding sites for beta1H, C3bINA, Factor B, and properdin. beta1H binding increased C3bINA binding 30-fold, while Factor B binding prevented C3bINA action on C3b and was competitive with beta1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the beta1H and C3bINA sites. Activators such as rabbit erythrocytes (E(R)) have previously been shown to interfere with the effectiveness of the control by C3bINA and beta1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of control does not occur on the surface of E(S), a nonactivator of the alternative pathway. On the basis of comparative binding studies, restriction of control is explained entirely by reduced binding of beta1H to E(R)C3b relative to E(S)C3b. Access of properdin, Factor B, C3bINA, and the Fab fragment of anti-C3 to the two cell types was unrestricted. Restriction of beta1H control could be generated on the surface of E(S) by removal of cell-surface sialic acid with neuraminidase (acylneuraminyl hydrolase; EC 3.2.1.18). This enzymatic treatment converted E(S) from a nonactivator to an activator of the alternative pathway.

Binding Sites↗
PubMed1990

Familial incidence of C3 nephritic factor, partial lipodystrophy and membranoproliferative glomerulonephritis.

C3 nephritic factor is an IgG autoantibody that causes complement activation by stabilizing the alternative pathway C3 convertase. It is associated with partial lipodystrophy and membrano-proliferative glomerulonephritis. The occurrence of C3 nephritic factor, partial lipodystrophy and membranoproliferative glomerulonephritis, whether singly or in any combination, is usually sporadic. We describe the coexistence of all three of these conditions in members spanning two generations of a single family. This suggests that the pathogenesis of these conditions may be linked and that genetically determined factors may, in some circumstances, contribute to disease susceptibility.

Biopsy↗
PubMed1986

Site-specific activation of the alternative pathway of complement. Synthesis of a hybrid molecule consisting of antibody and cobra venom factor.

Cobra venom factor (CoF) is an analog of the activated third component of human complement (C3b). Unlike C3b, CoF is completely resistant to inactivation by factors H and I, the endogenous control proteins of the alternative pathway of complement. This property makes CoF a useful reagent when unrestricted activation of complement is desired. CoF was covalently conjugated to the IgG fraction of rabbit antihuman erythrocyte antiserum, and the ability of the hybrid molecules to initiate activation of the alternative pathway at a specific site was tested. The hybrid molecules were heterogeneous polymers (CoFn-IgGn, where n is an integer greater than 1 for at least one of the components), and both components retained their individual functional activities. The alternative-pathway C3/C5 convertase complex was formed by adding purified factors B and D to the hybrid in the presence of magnesium. This complex (antibody-CoFBb) was able to bind to erythrocytes and activate both C3 and C5. Activation of C5 initiated formation of the potentially cytolytic membrane attack complex of complement on the surface of the cell. When used in combination with rabbit serum, the hybrid molecule was a potent mediator of complement-induced hemolysis. The decay kinetics of the hybrid C3/C5 convertase complex when bound to normal human erythrocytes were not first order, but 40% of the activity remained after 3.5 h at 37 degrees C. Conjugation of CoF to specific antibodies will permit investigation of the consequences of alternative-pathway activation at selective sites. These hybrids may also have therapeutic potential.

Complement Activating Enzymes↗
PubMed1977

The activation of the C3b feedback cycle with human complement components. II. Using components of the alternative pathway.

The C3 convertase of the C3b feedback cycle was generated in the fluid phase from C3b and factors B and D. It was shown to be an effective decomplementing agent when reacted with human serum. Many of the effects observed were dependent on the operation of the feedback during the ongoing reaction. Oxidization of factor B with iodine did not significantly increase the potency or the stability of the enzyme. Treatment of C3b with antrypol inhibited the formation of the enzyme. In preliminary experiments in the rat, the convertase was successfully formed in vivo, accompanied by cleavage of C3, consumption of C5, C6 and C7, and a biphasic change in the circulating neutrophils.

Animals↗
PubMed1991

Proposed structure of the F' allotype of human CR1. Loss of a C3b binding site may be associated with altered function.

Human CR1 is composed of tandem long homologous repeating (LHR) segments that encode separate binding sites for C3b or C4b. Homologous recombination with unequal crossover has been proposed as the genetic mechanism that gave rise to the CR1 alleles that differed in their total numbers of LHR. The F allotype has four LHR, named LHR-A, -B, -C, -D, 5' to 3'. The site in LHR-A preferentially binds C4b and those in LHR-B and -C prefer C3b. A previous study revealed the presence of a fifth LHR with sequences similar to LHR-B and a third C3b binding site in the S allotype of higher m.w. In the present study, an 18-kb EcoRV fragment that was associated with the expression of the lower m.w. F' allotype hybridized with a unique pattern of cDNA and intron probes specific for LHR-C. Deletion of LHR-B and one C3b binding site was proposed as the mechanism for the appearance of this F'-specific fragment. Functional differences among the CR1 variants were sought by comparative analyses of soluble rCR1 having one, two or three C3b binding sites. Although these three variants did not exhibit any significant differences in their capacities to act as cofactors for the cleavage of monomeric C3b, their relative affinities for dimeric ligand varied more than 100-fold. Furthermore, the variant with only one C3b binding site was at least 10-fold less effective in the inhibition of the alternative pathway C3 and C5 convertases. These observations suggested that the F' allotype may be impaired in its capacity to bind opsonized immune complexes, to inhibit the formation of the alternative pathway C3 and C5 convertases, and perhaps to mediate other CR1-dependent cellular responses.

Alleles↗
PubMed2003

Functional activity of the complement regulator encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma and certain B-cell lymphomas. The fourth open reading frame of the KSHV genome encodes a protein (KSHV complement control protein (KCP, previously termed ORF4)) predicted to have complement-regulating activity. Here, we show that soluble KCP strongly enhanced the decay of classical C3-convertase but not the alternative pathway C3-convertase, when compared with the host complement regulators: factor H, C4b-binding protein, and decay-accelerating factor. The equilibrium affinity constant (KD) of KCP for C3b and C4b was determined by surface plasmon resonance analysis to range between 0.47-10 microM and 0.025-6.1 microM, respectively, depending on NaCl concentration and cation presence. Soluble and cell-associated KCP acted as a cofactor for factor I (FI)-mediated cleavage of both C4b and C3b and induced the cleavage products C4d and iC3b, respectively. In the presence of KCP, FI further cleaved iC3b to C3d, which has never been described before as complement receptor 1 only mediates the production of C3dg by FI. KCP would enhance virus pathogenesis through evading complement attack, opsonization, and anaphylaxis but may also aid in targeting KSHV to one of its host reservoirs since C3d is a ligand for complement receptor 2 on B-cells.

Alternative Splicing↗
PubMed1985

Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system.

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol-reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.

Blood Platelets↗
PubMed1987

Activation of C5 by cobra venom factor is required in neutrophil-mediated lung injury in the rat.

Cobra venom factor (CVF)-induced systemic activation of the complement system in the rat has been shown to result in the development of acute lung microvascular injury and appearance in lungs and plasma of lipid peroxidation products. The pathogenesis of these events is dependent on complement and neutrophils and is sensitive to pretreatment of experimental animals with iron chelators or scavengers of hydroxyl radical. In order to further analyze the role of complement in the pathogenesis of acute lung injury in rats after systemic complement activation, two different CVFs have been employed in the present study. One was the previously used CVFn isolated from Naja n. naja venom, whereas the other factor, CVFh, was isolated from Naja h. haje venom. Both factors have been shown to activate the alternative complement pathway by forming a potent C3 convertase but differ with respect to their ability to bind and activate C5. CVFn but not CVFh activates C5 and distant complement components. When equal doses of C3-activating activity of CVFn or CVFh were injected intravenously into rats, CVFh-treated rats failed to develop acute lung injury, whereas CVFn-treated animals showed pronounced increases in lung vascular permeability. Similarly, in isolated blood perfused rat lungs neither the lung injury nor pulmonary hypertension caused by CVFn were found after injection of CVFh. In addition, CVFh-treated animals failed to show transient neutropenia or appearance in plasma of C5-derived chemotactic activity, although the extent of C3 conversion in vivo was identical to that seen in CVFn-treated rats. Morphologic examination of the lungs of the experimental animals revealed no signs of injury in CVFh-treated rats, whereas the lungs from CVFn-treated animals revealed interstitial and alveolar edema, as well as plugging of pulmonary capillaries with neutrophils, blebbing and/or destruction of vascular endothelial cells, fibrin deposition, and hemorrhage. These studies provide evidence that activation of the complement system involving C3 but not extending further in the complement sequence is not sufficient to bring about acute injury of the lung microvasculature and that the activation sequence must at least also involve C5.

Animals↗
PubMed1975

Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase.

A function of P in the alternative complement pathway is to prolong the first order decay of the hemolytic sites on EAC43B in a dose-dependent manner. As the number of initial convertase sites is not changed, even when activated properdin (P) increases the t1/2 10-fold or more, P acts to stabilize rather than to uncover additional sites. P binds to EAC43 to generate EAC43P in a reaction that proceeds slightly more rapidly at 15 degrees C than at 0 degrees C, but reaches the same plateau and does not require divalent cations. The presence of P on EAC43P not only stabilizes the convertase subsequently formed on that cell, but, alternatively, permits transfer to convertase sites on other cells with the stability of the recipient intermediate being dependent on the P available for transfer. The capacity of P to bind to C3b and stabilize C3B contrasts with the inhibitory effect of the C3b inactivator on formation of this amplification convertase.

Animals↗
PubMed1999

Renal, central nervous system and pancreatic overexpression of recombinant soluble Crry in transgenic mice. A novel means of protection from complement-mediated injury.

Crry is a potent complement regulator that inhibits classical and alternative pathway C3 convertases in rodents. We have produced transgenic animals expressing Crry as a recombinant soluble protein driven by the broadly active metallothionein-I promoter. These animals have high serum and urinary levels of rsCrry leading to inhibition of complement activity. In nephrotoxic serum nephritis (NSN), injected antibodies bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. We have shown that rsCrry can block such injury and reduce albuminuria by as much as 75%. Corresponding to the reduction in albuminuria was the complete absence of C3 staining in glomeruli by immunofluorescence microscopy in 17/20 transgene positive animals. Support for a local source of protective rsCrry in this model is provided by the demonstration of Crry transgene mRNA in the glomerulus and a very high fractional excretion of rsCrry in the urine. Therefore, rsCrry expression markedly ameliorates an antibody-induced disease model in vivo. In addition, local synthesis of Crry in other organs that are targets of immune injury has been found. For example, Crry transgene mRNA is present throughout the central nervous system and in pancreatic islets. Thus, continuous complement inhibition at the C3 convertase step appears to be feasible and is effective in complement-mediated injury states. A number of disease models affecting these target organs can be tested using these mice.

Animals↗
PubMed1976

The cofactors required by C3 nephritic factor to generate a C3 convertase in vitro.

The mechanisms by which a C3 convertase is generated by C3 nephritic factor (NeF) were investigated using purified NeF, C3, C3b, factor B and factor D of the alternative pathway of complement activation. NeF could generate a C3 convertase with C3 and B in the absence of D, and without cleavage of B. At lower concentrations of NeF the addition of D was required to generate a C3 convertase, and B cleavage now occurred. The generation of both the D-independent and D-dependent C3 convertases with NeF was inhibited by preincubation of the C3 source with C3b inactivator (KAF); isolated C3b was more efficient than the C3 preparations used in generating the D-independent C3 convertase with NeF. These experiments indicate that C3b is required for the formation of both convertases, and that the reaction occurring with apparently native C3 is due to trace amounts of C3b. It is concluded that the C3 convertase generated by NeF in the absence of D is C3bB (NeF), and that generated in the presence of D is the feedback convertase C3bBb. The relevance of these experiments to reactions which may occur in vivo is discussed.

Biological Products↗