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PubMed1999

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, C8alpha, C8beta, 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.

Amino Acid Sequence↗
PubMed2006

Complement activation in autoimmune demyelination: dual role in neuroinflammation and neuroprotection.

Multiple sclerosis and its animal model experimental allergic encephalomyelitis are inflammatory demyelinating diseases of the central nervous system mediated by activated lymphocytes, macrophages/microglia and the complement system. Complement activation and the C5b-9 terminal complex contribute to the pathogenesis of these diseases through its role to promote demyelination. C5b-9 was also shown to protect oligodendrocytes from apoptosis both in vitro and in vivo. Our findings indicate that activation of complement and C5b-9 assembly plays a pro-inflammatory role in the acute phase, but may also be neuroprotective.

Animals↗
PubMed1982

Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.

Complement C9↗
PubMed1996

C5b-8 step lysis of swine endothelial cells by human complement and functional feature of transfected CD59.

The authors established several swine endothelial cell (SEC) lines expressing human CD59 by transfection of cDNA, and assessed the function of the transfectant molecules in comparison with those of membrane cofactor protein (MCP) and decay-accelerating factor (DAF) in an in vitro hyperacute rejection model of swine to human discordant xenograft. At the usual expression rate, DAF and MCP protected SEC from human complement mediated cell lysis, but CD59 did not block human complement attack on SEC. However, CD59 protects SEC from cell lysis when sufficiently expressed as in human umbilical vein (HUVEC). The authors examined why CD59 needed so many molecules to protect human complement-mediated SEC lysis and found that SEC underwent lysis by human C5b-8. The degree of C5b-8 step lysis of SEC was approximately 70% of the total activation (C5b-9). Additionally, CD59 protected human complement activities less efficiently at the C5b-8 step than at the C9-step. Therefore, to overcome human complement mediated SEC lysis, C8 activity must be inhibited by dense expression of CD59.

Animals↗
PubMed2004

[Inhibitory effect of hDAF displayed on yeast cells on deposition of complement on the yeast cells].

AIM: To investigate whether the hDAF displayed on the surface of yeast cells can inhibit the deposition of C5b-9. METHODS: After treatment with normal human serum, deposition of human complement C5b-9 and DAF expression on yeast cells EBY100 were analyzed by flow cytometry. RESULTS: Yeast cells EBY100 could activate the complement molecules in human serum and lead to the deposition of C5b-9, while the deposition of C5b-9 on the surface of yeast cells with hDAF could significantly reduce the deposition of C5b-9. CONCLUSION: hDAF displayed on the surface of yeast cells can fold correctly and exhibit activity of complement's inhibition.

CD55 Antigens↗
PubMed1983

Interaction of the eighth component of guinea pig complement (C8) with the membrane-bound C5b-7 complex. The binding site of C8 to C5b-7 is formed on association of the alpha-gamma subunit with the beta-chain.

The eighth component (c8) of guinea pig complement consists of three polypeptide chains, the alpha-, beta-, and gamma-chains with M.W. of 60,000, 60,000, and 24,000, respectively. The alpha- and gamma-chains are bound by a disulfide bond(s) forming an alpha-gamma subunit, which is linked noncovalently to the beta-chain. The alpha-gamma subunit and the beta-chain were separated and purified from C8 by treatment with sodium dodecyl sulfate (SDS) and gel chromatography on Sephacryl S-300 in the presence of SDS. After removal of SDS, neither alpha-gamma nor beta showed the hemolytic activity of C8 when assayed independently, but showed significant activity in combination, indicating reconstitution of active C8. The recovery of hemolytic activity was 3.48%. When alpha-gamma and beta were incubated successively with EAC-7 with intervening reconstitution of active C8 on the cells was insignificant, irrespective of the order of the reactions. alpha-gamma and beta did not bind to EAC-7 when added separately, but after recombination 7% of alpha-gamma and 9% of beta bound to EAC-7 when EAC-7 was in excess. These results indicate that the binding site of guinea pig C8 to the membrane-bound C5b-7 complex does not exist on either alpha-gamma or beta only but stretches over both or is formed on one subunit after recombination of the subunits.

Animals↗
PubMed2005

Contrasting roles of complement activation and its regulation in membranous nephropathy.

The complement system is involved in defense against microorganisms, the processing of immune complexes and apoptotic debris, and the development of an appropriate immune response. Along with these physiologic effects, complement activation has the potential to result in tissue pathology. To limit this, various complement regulatory proteins (CRP) are present on host cells, including the glomerular podocyte. Experimental data from the Heymann nephritis (HN) rat model of human membranous nephropathy (MN) have shown that IgG antibodies in subepithelial immune deposits initiate complement activation and C5b-9-mediated damage of the overlying podocyte. Although IgG can activate the classical pathway, there also is evidence that alternative pathway activation occurs in MN, which could occur because of absent, dysfunctional, or inhibited podocyte CRP. Related to this are experimental data in HN showing the presence of antibodies that bind and inhibit podocyte CRP; although such antibodies have not been documented in human MN, a decrease in CR1 quantity on the podocyte has been observed. A s a result of a relative lack of CRP and the exposure of activating complement proteins to tubular cells, alternative complement pathway activation and C5b-9-mediated tubular injury can occur in MN and other proteinuric diseases. Overall, in a disease such as MN, the balance between complement regulation and activation is tipped toward its being activated. Therefore, a number of therapeutic approaches have been developed to counteract this, including recombinant forms of endogenous CRP and complement-inhibitory monoclonal antibodies. There is good reason to be optimistic that approaches to block complement activation will become viable therapy for human MN in the future.

Animals↗
PubMed2002

Expression of complement regulating factors in gastric cancer cells.

AIMS: To investigate the deposition of complement components, C3d and C5b-9, and the expression of complement regulating factors (S protein, membrane cofactor protein (MCP; CD46), protectin (CD59), decay accelerating factor (DAF; CD55), and type 1 complement receptor (CR1; CD35)) in gastric cancers. METHODS: Specimens of gastric cancer were examined by immunohistochemistry and immunoelectron microscopy. RESULTS: Four complement regulating factors (S protein, MCP, protectin, and DAF) were expressed on gastric cancer cells, in ultrastructurally localised areas on the cell membrane. CR1 was not expressed. The staining intensity of DAF in both differentiated and undifferentiated adenocarcinomas was significantly higher than in histologically normal gastric epithelium. Furthermore, the staining intensity of DAF in gastric cancers showing a diffusely infiltrating growth pattern was higher than in gastric cancers showing an expanding growth pattern. CONCLUSIONS: These data indicate that DAF may play a role in cancer cell infiltration and resistance in tumour cells.

Adenocarcinoma↗
PubMed1987

Activation of terminal components of complement in patients with Guillain-Barré syndrome and other demyelinating neuropathies.

In the present study, the role of antiperipheral nerve myelin antibody (anti-PNM Ab) in demyelination by generating the terminal attack complex (C5b-9) of complement was explored in patients with Guillain-Barré syndrome (GBS) and other demyelinating neuropathies. The presence in serum of SC5b-9, an inactive C5b-9 containing S protein, was assessed quantitatively by enzyme-linked immunosorbent assay using an antibody (Ab) to neoantigens expressed on C9 when complexed with C5b-8 or after tubular polymerization. SC5b-9 was detected in all 19 GBS, four patients with paraprotein-associated neuropathy and five of six patients with chronic recurrent polyneuritis. No SC5b-9 was detected in 10 normal controls. Kinetic studies from six GBS patients showed the highest values of SC5b-9 on the 3rd to 5th d of admission; in contrast, the anti-PNM Ab were highest on the day of admission. Anti-PNM Ab fell rapidly to very low levels by the 15th to 20th d. SC5b-9 declined with similar kinetics to undetectable levels by the 30th d. Levels of Ab and SC5b-9 did not quantitatively correlate with soluble immune complexes in these patients' serum. Membrane-bound C5b-9 was also detected by immunohistochemistry in the peripheral nerves from a GBS patient. These results, which show a relationship between levels of complement-fixing anti-PNM Ab and the tissue-damaging C5b-9 complex, suggest that peripheral nerve myelin may serve as the target for Ab-mediated complement attack.

Antigen-Antibody Complex↗
PubMedDate not supplied

Oligodendrocyte cell death in pathogenesis of multiple sclerosis: Protection of oligodendrocytes from apoptosis by complement.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. It is mediated by activated lymphocytes, macrophages, microglia, and complement. In MS, myelin-forming oligodendrocytes (OLGs) are the targets of inflammatory and immune attacks. OLG death by apoptosis or necrosis causes the cell loss seen in MS plaques. Studies of experimental allergic encephalomyelitis (EAE) in caspase 11-deficient mice show that caspase-mediated death of OLGs is critical to demyelination. Complement activation may affect MS pathogenesis through activated terminal complex C5b-9, which promotes demyelination, and through sublytic C5b-9, which protects OLGs from apoptosis. By inducing EAE in C5-deficient mice, we showed that complement C5 promotes axon preservation and new myelin formation, which protect OLGs from apoptosis. These findings indicate that activated complement C5b-9 plays a proinflammatory role in acute MS but may also protect OLGs from death in chronic MS.

Animals↗
PubMed1993

Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9.

Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.

Clusterin↗
PubMed1996

Isolation of the C9b fragment of human complement component C9 using urea in the absence of detergents.

The bactericidal activity of the C5b-9 complex of complement is dependent upon the terminal complement component C9. The precursor C5b-8 complex is not harmful to bacterial cells until C9 is added to complete the C5b-9 complex. The C9 molecule can be proteolytically cleaved by thrombin to yield an intact, nicked molecule that remains fully functional when added to either bacterial cells or erythrocytes bearing pre-formed C5b-8 complexes. In investigating the membranolytic function of C9 in the C5b-9 complex, the carboxyl-terminal portion of the nicked molecule (C9b) has been shown to be membranolytic when added to erythrocytes, liposomes, or bacterial inner membranes in the absence of any other complement components. The isolation of C9b from nicked C9 has been accomplished by preparative gel electrophoresis using detergents, however the study of the activity of C9b in membrane systems may be complicated by the possible presence of residual detergent. To address this concern, we have used 4 M urea in conjunction with hydroxyapatite chromatography and a phosphate elution procedure to separate the domains of nicked C9. The isolated C9b domain, free of detergents and in the absence of any other complement components, was found to be membranolytic. C9b isolated in this manner was capable of lysing erythrocytes and inhibiting the growth of bacterial spheroplasts.

Complement C9↗
PubMed1978

Molecular nature of the complement lesion.

The principle molecular event leading to membrane perturbation by complement is the assembly of the terminal five serum complement components (C5b-C9) into a macromolecular C5b-9 complex on the target membrane [Müller-Eberhard, H.-J. (1975) Ann. Rev. Biochem. 44, 697--723]. The present communication reports on the ability of purified C5b-9 complexes isolated from target membranes to become reincorporated into artificial lipid vesicles. The data indicate that the complex is a vertically oriented, hollow, cylindrical macromolecule possessing lipid-binding regions that enable one terminus to penetrate into the lipid bilayer. A transmembrane pore appears to be created at the attachment site of the C5b-9 complex.

Animals↗
PubMed1978

Complement lysis: the ultrastructure and orientation of the C5b-9 complex on target sheep erythrocyte membranes.

The C5b-9 complex derived from human serum and assembled on target sheep erythrocyte membranes is a thin-walled cylinder rimmed by an annulus at one end. The total height of the cylinder is 150 A, towards which the annulus contributes 30 A. The cylinder has an apparently uniform internal diameter of 100 A. The external diameter of the annulus is 200 A. The classical complement 'rings' visualized on membranes after complement lysis represent such C5b-9 cylinders perpendicularly oriented on the membranes. The thin-walled cylinder is anchored in the membrane matrix and the annulus located in the exterior membrane glycocalyx. At the sites of attachment of the C5b-9 complexes, the continuity of the membrane bilayer is disturbed and the presence of trans-membrane pores is indicated. The data essentially support the 'doughnut' theory of complement lysis.

Animals↗
PubMed1990

Regulatory control of the terminal complement proteins at the surface of human endothelial cells: neutralization of a C5b-9 inhibitor by antibody to CD59.

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.

Antibodies, Monoclonal↗