Interaction of complement proteins C5b-6 and C5b-7 with phospholipid vesicles: effects of phospholipid structural features

Complement components C5b-6 and C7 assemble to form C5b-7, which then interacts with membranes and commits the membrane attack complex to a target site. This protein-membrane association event was investigated to determine possible structural features that could contribute to a selective membrane attack. This system may also suggest general properties of protein-membrane insertion events. Initial binding of C5b-6 to membranes could potentially determine the site of assembly. However, binding of C5b-6 to membranes required phosphatidylglycerol or phosphatidic acid produced from egg phosphatidylcholine while binding of C5b-6 to phosphatidylcholine, phosphatidylserine, or phosphatidylinositol was undetectable. Binding to phosphatidic acid was irreversible, and the bound C5b-6 could no longer interact with C7. In contrast, C5b-7 interacted with all phospholipids tested. The rate-limiting process was the interaction of C5b-6 and C7, which displayed bimolecular properties and an activation energy of 37 kcal/mol. The C5b-7 complex showed 20-fold selectivity for small unilamellar phospholipid vesicles over large unilamellar vesicles. Vesicles carrying high negative charge densities were selected over neutral vesicles by a factor of about 5. Vesicles formed from phospholipids with short, saturated hydrocarbon side chains (dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine) were about 5-fold less effective than those formed from phospholipids with natural fatty acid distributions. The gel vs. fluid state had little influence on C5b-7 insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of human beta-endorphin with the terminal SC5b-9 and "preterminal" SC5b-7 and SC5b-8 complexes of human complement

Human beta-endorphin (beta H-EP) is demonstrated to bind to the "preterminal" SC5b-7 and SC5b-8 complexes and to the terminal SC5b-9 complex of human complement. Detailed binding studies revealed saturability, reversibility and structural specificity of the beta H-EP interaction with high or low affinity non-opiate binding sites on SC5b-7 and SC5b-9 complexes. The high affinity binding sites seem to be located predominantly on C5b, C6 or C7 subunits of the complexes.     

Elimination of terminal complement intermediates from the plasma membrane of nucleated cells: the rate of disappearance differs for cells carrying C5b-7 or C5b-8 or a mixture of C5b-8 with a limited number of C5b-9

We have previously shown that multiple complement (C) channels are required for lysis of a nucleated cell in contrast to the single channel requirement for erythrocytes. To further investigate this multichannel requirement for nucleated cells, we examined the stability of terminal C complexes in the plasma membrane of Ehrlich ascites tumor cells. Ehrlich cells bearing C5b-7 or C5b-8 with or without C9 were incubated at 37 degrees C or 0 degree C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. C5b-7, C5b-8, and C5b-8 in the presence of a limited number of C5b-9 complexes disappeared functionally from the plasma membrane at 37 degrees C, with initial half-lives of 31, 20, and 10 min, respectively. Disappearance of these complexes did not occur at 0 degree C, nor did disappearance occur at 37 degrees C when formed on sheep erythrocytes. The fate of C5b-8 complexes on the surface of Ehrlich cells was traced with colloidal gold particles bound to C5 determinants on C5b-8 with the use of immunoelectron microscopy. Colloidal gold could be seen on the cell surface after specific binding to cells carrying C5b-8 sites at 0 degree C. After incubating these cells at 37 degrees C, gold particles were internalized into the cell continuously via endocytic vesicles. It is postulated that terminal C complexes may stimulate or accelerate the removal of these complexes from the cell surface.     

Interaction of Human Waldenström's IgM Proteins with Guinea Pig and Human Complement1

The activity of purified human Waldenström's IgM proteins to fix complement of human and guinea pig origins was compared at different temperatures using the polystyrene latex particle-adsorption method. It was shown that the interaction of the IgM proteins with complement differed depending on the source of complement and that a pronounced heterogeneity in complement-fixing activity was observed among the IgM proteins when tested with guinea pig complement. Thus, by the use of guinea pig complement, six human IgM proteins examined were classified roughly into two groups, one having a high and the other a low activity at 3 C as well as at 37 C. With human complement, five proteins showed a rather uniform activity at 37 C. However, there was one protein with no detectable activity, suggesting the presence of non-complement-fixing protein in the IgM class. All the six proteins showed no significant activity with human complement at 3 C. No antigenic difference has been found as yet in the Fc or Cμ2 region among these IgM proteins examined.     

Complement component 5a (C5a)

The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.     

The structure of human complement component C7 and the C5b-7 complex

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.     

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.     

Phylogenetic Analysis of the Homologous Proteins of the Terminal Complement Complex Supports the Emergence of C6 and C7 Followed by C8 and C9

The plasma complement system comprises several activation pathways that share a common terminal route involving the assembly of the terminal complement complex (TCC), formed by C5b–C9. The order of emergence of the homologous components of TCC (C6, C7, C8α, C8β, and C9) has been determined by phylogenetic analyses of their amino acid sequences. Using all the sequence data available for C6–C9 proteins, as well as for perforins, the results suggested that these TCC components originated from a single ancestral gene and that C6 and C7 were the earliest to emerge. Our evidence supports the notion that the ancestral gene had a complex modular composition. A series of gene duplications in combination with a tendency to lose modules resulted in successive complement proteins with decreasing modular complexity. C9 and perforin apparently are the result of different selective conditions to acquire pore-forming function. Thus C9 and perforin are examples of evolutionary parallelism. Received: 16 August 1998 / Accepted: 12 March 1999     

Solubilization of the Membrane-Bound Sialidase from Pig Brain by Treatment with Bacterial Phosphatidylinositol Phospholipase C

The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5'-nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37 degrees C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5'-nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 x 10(-4) and 0.98 x 10(-4) M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4 degrees C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.     

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

补体第六成分（C6）在汉族5个群体中的多态分布

用聚丙烯酰胺等电聚焦技术和免疫酶标法,调查分析了汉族5个群体补体第六成分(C6)的遗传多态性,得出基因频率如下。郑州汉族:C6*A 0.4521、C6*B 0.5228、C6*B_2 0.0183、和C6*R 0.0068。兰州汉族:C6*A 0.4612、C6*B 0.5218和C6*B_2 0.0170。呼和浩特汉族:C6*A 0.4452、C6*B 0.5286、C6*B_2 0.0214和C6*R 0.0048。西安汉族:C6*A 0.4899、C6*B 0.4874、C6*B_2 0.0126和C6*R 0.0101。广东梅州客家人:C6*A 0.4569、C6*B 0.5152和C6*B_2 0.0279。C6*R为罕见等位基因之频率。     

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.     

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.     

Prevention of Arthritis by Locally Synthesized Recombinant Antibody Neutralizing Complement Component C5

Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production.     

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.     

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.