Fluid-phase assembly of the membrane attack complex of complement

The dynamics and protein stoichiometry of the fluid-phase assembly of the membrane attack complex of complement were characterized by using light-scattering intensity measurements. The assembly proceeded in an ordered manner with generation of stable and highly reproducible intermediates. In the absence of phospholipid or C8, mixtures of C5b-6 and C7 self-associated to fluid phase-C5b-7 which had a weight-average molecular weight of (4.1 +/- 0.2) X 10(6). This corresponded to an average of nine C5b-7 complexes per particle. The particles appeared heterodisperse on sucrose gradients with S20,W values ranging from 21 to 39 S. Addition of C8 and C9 caused no further aggregation or disassembly of the particles. When excess C8 was added to the aggregated C5b-7, the ratio of C8 incorporated per C5b-7 moiety was 0.98 +/- 0.03. At saturating levels of C9, the C9/C5b-8 ratio in the particles was 7.2 +/- 0.6. Incorporation of C8 caused a small increase in the Z-averaged particle diffusion coefficient [(9.9-10.3) X 10(-8) cm2/s], indicating that it added in a manner that "filled in the gaps" in the C5b-7 particles. C9 caused only small decreases in the particle diffusion coefficient and substantially decreased the f/fmin ratio. The time course for C9 incorporation into fluid phase-C5b-8 indicated an initial rapid phase followed by a slow phase. The rapid phase corresponded to the incorporation of about one C9 for every two C5b-8 complexes. This suggested that one C9 binding site was accessible on about half of the C5b-8 complexes. This may imply that only about half of the C5b-8 complexes were capable of C9 polymerization so that the ratio of C9 incorporated per functional C5b-8 was (14 +/- 2)/1. The initial velocity of the slow phase of C9 addition gave an activation energy of 37 kcal/mol. The activation energy for C5b-8-independent polymerization of C9 had a similar value of 41 kcal/mol. Light-scattering intensity measurements seemed to be a highly reliable method for quantitative characterization of the fluid-phase assembly.     

Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles

Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of less than 1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 +/- 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 +/- 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of polymerization of a fluoresceinated derivative of complement protein C9 by the membrane-bound complex of complement proteins C5b-8

The fluorescence self-quenching by energy transfer of FITC-C9, a fluoresceinated derivative of human complement protein C9 [Sims, P.J. (1984) Biochemistry (preceding paper in this issue)], has been used to monitor the kinetics of C9 polymerization induced by the membrane-associated complex of complement proteins C5b-8. Time-based measurements of the fluorescence change observed during incubation of FITC-C9 with C5b-8-treated sheep red blood cell ghost membranes at various temperatures revealed that C9 polymerization induced by the C5b-8 proteins exhibits a temperature dependence similar to that previously reported for the complement-mediated hemolysis of these cells, with an Arrhenius activation energy for FITC-C9 polymerization of 13.3 +/- 3.2 kcal mol-1 (mean +/- 2 SD). Similar measurements obtained with C5b-8-treated unilamellar vesicles composed of either egg yolk phosphatidylcholine (egg PC), dipalmitoylphosphatidylcholine (DPPC), or dimyristoylphosphatidylcholine (DMPC) revealed activation energies of between 20 and 25 kcal mol-1 for FITC-C9 polymerization by C5b-8 bound to these membranes. Temperature-dependent rates of C9 polymerization were observed to be largely unaffected by the phase state of membrane lipid in the target C5b-8 vesicles. The significance of these observations of the mechanism of C9 activation of membrane insertion is considered.     

Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.     

Formation of ion-conducting channels by the membrane attack complex proteins of complement.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.     

The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9.

A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.     

Molecular composition of the terminal membrane and fluid-phase C5b-9 complexes of rabbit complement. Absence of disulphide-bonded C9 dimers in the membrane complex

The terminal membrane C5b-9(m) and fluid-phase SC5b-9 complexes of rabbit complement were isolated from target sheep erythrocyte membranes and from inulin-activated rabbit serum respectively. In the electron microscope, rabbit C5b-9(m) was observed as a hollow protein cylinder, a structure identical with that of human C5b-9(m). Monodispersed rabbit C5b-9(m) exhibited an apparent sedimentation coefficient of 29 S in deoxycholate-containing sucrose density gradients, corresponding to a composite protein-detergent molecular-weight of approx. 1.4 X 10(6). Protein subunits corresponding to human C5b-C9 were found on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. By densitometry, there were consistently six molecules of monomeric C9 present for each monomeric C5b-8 complex. Fluid-phase rabbit SC5b-9 was a hydrophilic 23 S ma macromolecule that differed in subunit composition from its membrane counterpart in that it contained S-protein and only two to three molecules of C9 per monomer complex. The data are in accord with the previous report on human C5b-9 that C5b-9(m) contains more C9 molecules than SC5b-9 [Ware & Kolb (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6426-6430]. They corroborate the previous molecular-weight estimate of approx. 10(6) for C5b-9(m) and thus support the concept that the fully assembled, unit lesion of complement is a C5b-9 monomer [Bhakdi & Tranum-Jensen (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1818-1822]. They also show that C9 dimer formation is not required for assembly of the rabbit C5b-9(m) protein cylinder, or for expression of its membrane-damaging function.     

On the cause and nature of C9-related heterogeneity of terminal complement complexes generated on target erythrocytes through the action of whole serum

The binding of C8 and C9 from human serum to target erythrocytes was quantified, and the molecular stoichiometries of C9:C8 within terminal C5b-9(m) complexes were determined. Low doses of serum generated terminal complexes with mean C9:C8 ratios of 2 to 3:1, whereas complexes generated by highest serum doses harbored an average of six to eight C9/C8 molecules. From the collective biochemical and ultrastructural data, we concluded that heterogeneous populations of terminal complexes regularly form on target membranes; those containing high numbers of C9 molecules (greater than or equal to six to eight) exhibit the structure of the classical "lesion", whereas those containing low numbers of C9 do not exhibit this typical structure, although they probably still function as small pores. A major cause for this heterogeneity of the lesions derives from shortage of C9, which is naturally present in a 2 to 1 molar ratio relative to C8 in serum. Generation of terminal complexes harboring high numbers of C9 on erythrocyte membranes is possible in spite of this natural shortage because SC5b-9 does not form in the fluid phase to compete for C9 binding. If interrupted, the process of C9-C9 oligomerization cannot be recontinued, and "incomplete" C5b-9 complexes are unable to bind additional C9 upon reincubation with this component. The demonstrated heterogeneity of terminal complexes with respect to their C9 content may explain the functional heterogeneity of complement lesions observed previously by other investigators.     

Inhibition of homologous complement by CD59 is mediated by a species-selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9.

The capacity of the human complement regulatory protein CD59 to interact with terminal complement proteins in a species-selective manner was examined. When incorporated into chicken E, CD59 (purified from human E membranes) inhibited the cytolytic activity of the C5b-9 complex in a manner dependent on the species of origin of C8 and C9. Inhibition of C5b-9-mediated hemolysis was maximal when C8 and C9 were derived from human (hu) or baboon serum. By contrast, CD59 showed reduced activity when C8 and C9 were derived from dog or sheep serum, and no activity when C8 and C9 were derived from either rabbit or guinea pig (gp) serum. Similar specificity on the basis of the species of origin of C8 and C9 was also observed for CD59 endogenous to the human E membrane, using functionally blocking antibody against this cell surface protein to selectively abrogate its C5b-9-inhibitory activity. When E bearing human CD59 were exposed to C5b-8hu, CD59 was found to inhibit C5b-9-mediated lysis, regardless of the species of origin of C9, suggesting that the inhibitory function of CD59 can be mediated through recognition of species-specific domains expressed by human C8. Consistent with this interpretation, CD59 was found to bind to C5b-8hu but not to C5b67hu or C5b67huC8gp. Although CD59 failed to inhibit hemolysis mediated by C5b67huC8gpC9gp, its inhibitory function was observed for C5b67huC8gpC9hu, suggesting that, in addition to its interaction with C5b-8hu, CD59 also interacts in a species-selective manner with C9hu incorporated into C5b-9. Consistent with this interpretation, CD59 was found to bind both C5b67huC8gpC9hu and C5b-8huC9gp, but not C5b67huC8gpC9gp. Taken together, these data suggest that the capacity of CD59 to restrict the hemolytic activity of human serum complement involves a species-selective interaction of CD59, which involves binding to both the C8 and C9 components of the membrane attack complex. Although CD59 expresses selectivity for C8 and C9 of human origin, this "homologous restriction" is not absolute, and this human complement regulatory protein retains functional activity toward C8 and C9 of some nonprimate species.     

Multimeric C9 within C5b-9 deposits in unique locations in the cell wall of Salmonella typhimurium

We have shown previously that multimeric C9 within C5b-9 (C9:C5b-8 greater than 3:1) is needed for killing of a rough strain of Escherichia coli. We now extend these studies using serum sensitive, rough (R) and serum resistant, wild type (WT) strains of Salmonella typhimurium as well as a mutant S. typhimurium strain (TS) with a temperature sensitive mutation in synthesis of keto-deoxy-octulosonate, a constituent within the deep core structure of Salmonella LPS. Both R and TS required multimeric C9 within C5b-9 to be killed. Addition at 37 degrees C of increasing inputs of C9 to TS or R bearing C5b-9 led to a dose-related increase in C9 binding and killing. In contrast, addition of high inputs of C9 to the same strains at 4 degrees C, a procedure that limits the C9:C5b-8 ratio to 1:1, resulted in low C9 binding and minimal killing. Bactericidal C5b-9 formed at 37 degrees C on R and TS with high inputs of C9 co-sedimented with the bacterial outer membrane on sucrose density gradient analysis. Non-bactericidal C5b-9 on R, WT, and TS co-sedimented near the inner membrane, despite the presumed lack of association between these constituents. Whereas 125I C9 within the non-bactericidal pools immunoprecipitate with anti-C5, 125I C9 within bactericidal pools did not immunoprecipitate with anti-C5, anti-C7, or anti-C9. These findings suggest that bactericidal C5b-9 may be deposited in a unique location or configuration within the bacterial cell wall.     

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.     

Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.     

Relative inefficiency of terminal complement activation

The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.     

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.     

The C5b-9 complex: subunit composition of the classical and alternative pathway-generated complex.

The membrane attack complex of complement (C5b-9) is identical in composition regardless of which pathway of activation was instrumental in its formation. Band V protein was consistently a subunit of the soluble complex. Since band V protein is not required for complement-dependent cytolysis, it probably represents a membrane site equivalent in serum of the nascent C5b-9 complex.     

Complement lysis of U937, a nucleated mammalian cell line in the absence of C9: effect of C9 on C5b-8 mediated cell lysis

Previous studies have demonstrated that in general, nucleated cells are more resistant to killing by serum complement than are erythrocytes. During studies aimed at defining the mechanisms of nucleated cell resistance, we found that the human histiocytic cell line U937 was easily lysed by homologous serum. U937 cells were also killed by serum depleted of C9, but not by serum depleted of C8, implying that the C5b-8 complex was sufficient to cause lysis of these cells. Enumeration of complexes on the cell surface demonstrated that approximately 40-fold more complexes were required to lyse U937 cells in the absence of C9 than in the presence of an excess of C9. Examination of the effects of small amounts of C9 on lysis of U937 cells by the C5b-8 complex demonstrated that at very low doses, C9 inhibited C5b-8 mediated lysis. The use of radiolabeled anti-C8 antibody showed that C5b-8 complexes were eliminated from the surface of U937 cells at 37 degrees C, and C9 at the dose causing inhibition of lysis accelerated the elimination of complexes. These results suggest that the increased lytic potential resulting from binding of small amounts of C9 to C5b-8 complexes is outweighed by enhanced elimination of complexes resulting in decreased cell death.     

The SC5b-7 complex: formation, isolation, properties, and subunit composition.

Activation of C in C8-depleted serum results in the formation of a soluble complex containing C5, C6, and C7. The complex has an electrophoretic mobility of an alpha-globulin, an s-rate of 18.5S, and a m.w. of 668,000 daltons. This complex was isolated and upon SDS polyacrylamide gel electrophoresis it was found to contain, in addition to C5b, C6 and C7, an 88,000 dalton glycoprotein. The protein was identified as the band V protein of the soluble C5b-9 complex. It is referred to as SIIIs-protein, or S-protein. Since the S-protein does not bind to C5b-6, it is concluded that it is incorporated during the fusion of C5b-6 with C7. The SC5b-7 complex exhibits the same neoantigen as the SC5b-9 complex, but compared to the C5b-6 complex it appears to contain an additionally qualitatively distinct neoantigen.     

Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.     

Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group

S protein, an inhibitor to the membrane attack complex of complement, was purified from human plasma. The procedure involved barium citrate adsorption and fractionation by poly(ethylene glycol) 4000 precipitation, followed by chromatography on DEAE-Sephacel, Blue Sepharose, Sephacryl S-200, and finally anti-albumin-Sepharose. Reduced glutathione was added throughout to inhibit spontaneous formation of disulfide-linked S-protein dimers. The recovery was 7%, resulting in approximately 10 mg of pure S protein from 1 L of starting plasma. S protein is a single-chain molecule; sedimentation equilibrium ultracentrifugation yielded a molecular weight of 83 000; the s020,W value was estimated to be 4.0 S. The purified protein contained a free, reactive thiol group causing spontaneous formation of disulfide-linked S-protein dimers. Alkylated and nonalkylated S proteins were equally active in inhibiting C9 polymerization, catalyzed by the C5b-8 complex. In parallel with the inhibition of C9 polymerization, nonalkylated S protein catalyzed the formation of disulfide-linked C9 dimers, presumably through disulfide interchanges.     

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified . Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.