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PubMed1981

Proteolysis of the monomeric and dimeric C5b-9 complexes of complement: alteration in the susceptibility to proteases of the C9 subunits associated with C5b-9 dimerization.

The C5b-9 monomer having the sedimentation coefficient of 23S was extracted from the rabbit erythrocyte membranes that had been treated with a limiting amount of C9-deficient human serum and of 125I-C9. Upon proteolysis by trypsin and chymotrypsin, the C9 subunits of this complex were cleaved by these enzymes at multiple sites, yielding fragments with m.w. ranging fro 40,000 to 19,000. The uncomplexed C9 was also cleaved by both enzymes at multiple sites. By contrast, the C9 subunits of the C5b-9 dimer were found to be totally insusceptible to chymotrypsin under the conditions studied (37 degrees C; 24 hr) and only partially susceptible to trypsin (33% of the C9 subunits were cleaved by trypsin into 2 fragments during incubation at 37 degrees C for up to 24 hr). Therefore, these results indicate that, although the binding of C9 molecules to the C5b-8 complex (C5b-9 monomer formation) does not significantly affect the susceptibility to proteases of the C9 molecules, C5b-9 dimer formation markedly limits the accessibility of proteases to the C9 subunit molecules. A implication of this finding to a role for C9 in C5b-9 dimerization is discussed.

Animals↗
PubMed1981

The membrane attack mechanism of complement: photolabeling reveals insertion of terminal proteins into target membrane.

We have utilized a membrane-restricted, photoactivable glycolipid probe to investigate the protein-lipid interactions involved in complement (C) mediated lysis of a target membrane. The purified C proteins C5b-6, C7, C8, and C9 were added to artificial membrane vesicles containing the 14C-labeled photoreactive probe anchored in the outer monolayer of the membrane, and 6-carboxyfluorescein trapped in the lumen as an indicator for effective lysis. Irradiation of the membrane samples at different stages of functional complex assembly resulted in labeling of each of the 5 terminal C proteins, indicating that all 5 proteins become inserted into the hydrophobic milieu of the membrane during some stage of complex assembly. However, at the final stage of complex assembly, only C9 appeared to be labeled. Because we can demonstrate that the photoreactive probe has no strong affinity for C9 over the other terminal components (C5b-C8), the extensive change in labeling specificity during assembly is evidence for substantial changes in protein-lipid and possibly protein-protein interactions during formation of the C lesion.

Cell Membrane↗
PubMed1980

Analysis of solute diffusion across the C5b-9 membrane lesion of complement: evidence that individual C5b-9 complexes do not function as discrete, uniform pores.

We have investigated the diffusion of radiolabeled nonelectrolytes across the membranes of resealed erythrocyte ghosts that had been treated with the terminal complement components C5b-9 and incubated under nonlytic steady state conditions. For all solutes tested, we note that diffusion across the C5b-9 lesion is retarded to rates more than 2 orders of magnitude slower than can be anticipated for a transmembrane diffusional channel of the dimensions suggested by the ultrastructure of the C5b-9 membrane lesion. Furthermore, direct measurement of the relative selectivity of the C5b-9 membrane lesions to permeation by solutes of differing molecular radii suggests that individual membrane lesions are not of uniform functional size. Data are presented that suggest that the functional heterogeneity of the membrane lesion is due to the aggregation of individual membrane bound C5b-9 complexes into larger functional units and not due to variable stoichiometry of C9 within the individual C5b-9 complex.

Cell Membrane Permeability↗
PubMed1978

The structural events associated with the attachment of complement components to cell membranes in reactive lysis.

Electron microscopic study of the events occurring at the cell membrane during reactive lysis by complement, showed that a foliaceous particle was formed at the C5b-7 stage, that enlarged to a particle with a variable number of arms at the C5b-8 stage. Up to this point, no typical complement lesions were found. At the C5b-9 stages, the particles were completely converted to typical complement lesions, i.e. hollow cylinders projecting from the cell membrane and partly penetrating it. C5b-9 complexes assembled in the fluid phase did not show the typical structure of the lesions, but were amorphous masses of fibres.

Complement C5↗
PubMed1979

In vitro activation of complement by isolated human heart subcellular membranes.

Activation of human complement (C) occurred in vitro when mitochondrial membranes isolated from normal human heart tissue were incubated with normal human serum. This activation, as measured by C3 depletion, was not completely inhibited by blocking classical pathway activity in serum treated with EGTA, in C2-deficient serum, or in C1-depleted serum, nor in serum heated at 50 degrees C for 30 min to block the alternative pathway, but it could be prevented by blocking the classical and the alternative pathway simultaneously with EDTA, or by treating heated serum (50 degrees C. 30 min) with EGTA. Factor B was converted in normal serum as well as in EGTA-treated serum, but not in EDTA-treated serum. Mitochondrial membranes had no direct enzymatic or other activity that could inactivate functionally or highly purified C4 or C3, but the membranes could bind and activate C1 either in serum or in functionally pure C1 preparations. C4 also bound to the mitochondrial membranes only in the presence of C1. These data suggest that the activation of C by heart subcellular membranes involved both the classical and the alternative pathways, that the mitochondrial membrane preparations were capable of forming stabel complexes with C1 and C4, but not C3, and that the mitochondrial membrane preparations did not contain enzymes or have inherent properties that could directly cause C3 conversion.

Adsorption↗
PubMed1995

Complement component C7 is a plasminogen-binding protein.

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.

Autoradiography↗
PubMed1976

Potentiation of C56-initiated lysis by leucocyte cationic proteins, myelin basic proteins and lysine-rich histones.

Synthetic polycations such as poly-L-lysine (PLL) have recently been shown to enhance C56-initiated lysis by neutralization of serum-derived inhibitors of the C567 complex, collectively designated C567-INH. In the present report we have examined the effect of several naturally occurring polycations on C56-initiated lysis. Lysosomal granule extracts from rabbit peritoneal exudate cells were found to potentiate C56-initiated lysis via counteraction of C567-INH in the fluid phase; this was dependent upon the amount of C567-INH present and independent of cell concentration. The basic proteins of guinea-pig, bovine, and monkey myelin as well as lysine-rich histones also potentiated EC567 formation, but this effect seemed to occur predominantly at the cell surface. The presence of biologically derived cationic proteins at sites of complement activation during inflammation thus might lead to enhanced tissue damage by favouring the formation of cell-C567 intermediates by either or both of these mechanisms.

Animals↗
PubMed1977

C567-initiated cytolysis of lymphoid cells: description of the phenomenon and studies on its control by C567 inhibitors.

Cells of the Raji human lymphoblastoid line, when pretreated with the metabolic inhibitor puromycin were found to be susceptible to killing by the isolated proteins of the complement attack mechanism (C5-9). Incubation of 51Cr-labeled lymphoblastoid cells with purified C56 and C7 resulted in the formation of a lymphoblast-C567 (LC567) intermediate, and the addition of purified human C8 and C9 resulted in release of 51Cr from these cells. Serum C567 inhibitors (C567-INH), purified human lipoproteins, and dextran sulfate, each previously shown to inhibit the attachment of the C567 trimolecular complex to erythrocytes, also inhibited the formation of LC567, and as a consequence, C56-initiated cytotoxicity; the polycation protamine sulfate counteracted the inhibitory effect of dextran sulfate. Thus, the potential for damage of bystander nucleated cells exists when C56 is generated in solution, and is influenced by agents known to modulate the hemolytic activity of C567. It is suggested that these mechanisms may be involved in the control of the function as well as the viability of various nucleated cells.

Cell Line↗
PubMed1977

Deviated lysis: transfer of complement lytic activity to unsensitized cells. IV. Parital isolation of the activity.

Deviated lysis (d.l.) was previously characterized as the lysis of non-sensitized erythrocytes by activated complement (C) in the presence of EDTA (1, 2, 3). The lytic activity was present in serum fractions of a m.w. in the proximity of 220,000. All the C factors C5 through C9 were found in these fractions and they were all needed for lysis. It is proposed that in d.l. small aggregates of the C components C5 through C9 coexist in the reaction mixture without further interaction. Only when appropriate receptors such as present on target cells surfaces are available, the factors react in a sequential order eventually to result in lysis of the target cell.

Animals↗
PubMed1977

Complement system in human colostrum: presence of nine complement components and factors of alternative pathway in human colostrum.

Evidence has been obtained for the presence in human colostrum of all nine components of complement (C), C1 through C9, and factors of the alternative pathway. Samples of colostrums collected from five women at 1-4 days after normal parturition were assayed for the haemolytic activities of individual components. As compared with normal human sera, the activities of each component ranged from 0.03 to 7% of those in sera. The activities of C4, C7 and C9 were relatively high, while that of C1 was extremely low. In most of the cases, the activities of individual components gradually increased following delivery, when expressed as the activity per unit weight (g) of protein in the colostrum. When the colostrums were treated with cobra venom factor, most of the colostrums showed 10-20% reduction in the C3 activity. This finding indicates the presence of factors such as B and D which are involved in the activation of C through the alternative pathway. The role as a defense factor of the C system in human colostrum and milk is discussed in connection with the ability of secretory IgA to react with C.

Colostrum↗
PubMed1976

A new activity of complement component C3: cell-bound C3b potentiates lysis of erythrocytes by C5b,6 and terminal components.

EAC4b,3b (sheep erythrocytes carrying rabbit antibody and guinea pig complement component fragments C4b and C3) adsorb human C5b,6 reversibly; the avidity of binding varies inversely with ionic strength. We believe that the receptor of C5b,6 is contributed by the cell-bound C3b because the binding capacity of EAC4b,3b varies with C3b multiplicity and can be blocked with rabbit antibody to guinea pig C3. The fixation of C5b,6 to the erythrocyte-bound C3b serves to concentrate C5b,6 on the cell surface; as a consequence, the hemolytic efficiency of C5b,6 is almost 100 times greater when assayed with EAC4b,3b than with plain erythrocytes. This potentiation represents a hitherto unrecognized function of cell-bound C3b.

Animals↗
PubMed1989

The release of C5a in complement-activated serum does not require C6.

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.

Animals↗
PubMed1983

Activation of the fifth and sixth components of the human complement system: C6-dependent cleavage of C5 in acid and the formation of a bimolecular lytic complex, C5b,6a.

Acidification of C5 and C6 or serum to pH 6.4 at 0 degrees C, followed by neutralization, generates a factor-designated C(56)a that causes lysis of nonsensitized erythrocytes in the presence of C7, C8, and C9. C(56)a is functionally similar to alternative pathway-generated C5b,6 in respect to the formation of C5b,6,7 sites on cells, the potentiation of lytic activity by membrane-bound C3b or the membrane-active agent A2C, and the required species compatibilities between target membranes and terminal components for optimal activity. The formation of C(56)a complex from purified components C5 and C6 proceeds independently of the classical or alternative pathway C5 convertases and requires the simultaneous H+ ion treatment of the components. The generation of C(56)a from C5 and C6 and the physicochemical properties of the complex were studied in detail and compared with those of C5b,6. Acid generation of C(56)a is dose-dependent on C5 and C6 and its efficiency is similar to that of the conventional convertase in the production of lytic activity. Sucrose gradient ultracentrifugation of C(56)a containing activated 125I-C5 demonstrated a shift in sedimentation from that of native C5 to 11S, which is consistent with C5,6 complex formation. C(56)a sedimentation was identical to C5b,6, and both migrated coincident with lytic complex activity. These complexes, however, are not identical because unlike C5b,6, C(56)a is unstable at 37 degrees C, demonstrating a nonlinear decay curve. In the presence of C7, both complexes exhibit similar first order decay with a T1/2 of 3 min at 37 degrees C. SDS-PAGE autoradiographic analysis of the C5-subunit structure of 125I-C5 in C(56)a and the Zx-activated C5b,6 complex prepared from purified components showed similar alpha-chain cleavage to several fragments of 109,000, 100,000, and 58,000 daltons. Conversion to lower m.w. peptides by acid treatment was more extensive. Comparison of the 125I-C5 polypeptide chains in the membrane attack complex extracted from guinea pig erythrocyte membranes, prepared by acid activation or classical pathway lysis with whole serum, demonstrated similar C5 alpha-chain cleavage to a predominant subunit of 102,000 daltons. Acid activation also produced a 109,000 dalton C5 alpha'-fragment barely detectable with classical pathway activation. Low pH treatment of C5 alone did not inactivate C5 function, form a lytic complex on the subsequent addition of C6, or cleave the C5 alpha-chain. Thus, it is postulated that local high H+ ion concentration during simultaneous acidification of C5 and C6 allows complex formation with the concomitant C6-dependent cleavage of the C5 alpha-chain and the generation of lytic capacity.

Centrifugation, Density Gradient↗
PubMed1977

Enhancement of C56-initiated lysis by cell-bound C3 fragments: evidence for a mechanism independent of the prior binding of C56 to C3b.

Cell-bound C3b can reversibly bind C56, the activated complex of the fifth (C5) and sixth (C6) components of complement, and in this way potentiate C56-initiated lysis by favoring the formation of C567 at the cell surface. We report here another way in which cell-bound C3 fragments can enhance C56-initiated lysis, which involves C567 generated in the fluid phase rather than at the cell surface. Evidence for the involvement of fluid phase C567 was obtained by use of dextran sulfate, which is known to inhibit the hemolysis of E mediated by fluid phase C567. Dextran sulfate strongly inhibited the formation of C567 sites on cells bearing C4b and C3b (EAC4b3b) as well as on unmodified E when C56 and C7 were added simultaneously to the cells. By contrast, dextran sulfate had virtually no effect on the reaction sequence involving the prior binding of C56 to C3b and subsequent formation of C567 at the cell surface. Treatment of EAC4b3b with either anti-C3 Fab' fragments or the C3b inactivator reduced but did not eliminate the enhancement of hemolysis, raising the possibilities that a C3 fragment(s) other than C3b also can enhance C56-initiated lysis and/or that the enhancement is indirect without a requirement for an interaction between C567 and the cell-bound C3 fragment itself.

Complement C3↗