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PubMed1983

Immunoradiometric assay for human complement component C9 utilising monoclonal antibodies.

A two-site immunoradiometric assay for human C9 has been developed. The assay utilised two non-competing monoclonal antibodies to C9 in a single incubation assay protocol. The detection limit of the assay was 0.1 ng (1.4 X 10(-15) moles) in a sample volume of 100 microliters. Using this assay the C9 concentration in normal human plasma was 60.2 +/- 14.9 mg/l (mean +/- 1 standard deviation, 8.5 X 10(-10) mol/l. Significantly elevated levels were found in the plasma of patients with rheumatoid arthritis (90.4 +/- 19.9 mg/l, mean +/- 1 SD). Measurements of C9 in cerebrospinal fluid and synovial fluid were also performed. The low levels of C9 in cerebrospinal fluid (less than 1 mg/l), undetectable by previously available assay methods, were easily measurable with this highly sensitive assay.

Antibodies, Monoclonal↗
PubMed1997

The administration of complement component C9 enhances the survival of neonatal rats with Escherichia coli sepsis.

To determine the significance of neonatal C9 deficiency, an animal model was developed in the rat. By rocket immunoelectrophoresis, the concentration of C9 in pooled adult rat serum was 224 +/- 7.2 microg/mL. In contrast, the concentration of C9 in pooled serum from 1-d-old rats was only 43 +/- 3.8 microg/mL and increased during the first 3 wk of life to 170 +/- 20 microg/mL. Similarly, the capacities of neonatal rat serum to kill two pathogenic strains of Escherichia coli and to lyse sensitized sheep erythrocytes were diminished compared with adult serum but increased during the first 3 wk of life. Supplemental human C9 significantly enhanced the bactericidal and hemolytic activity of neonatal rat serum. The capacity of neonatal rats to survive after the intrapulmonary injection of E. coli was positively correlated with the serum C9 concentration, bactericidal activity, and hemolytic activity. In 2-d-old rats infected with E. coli, the intraperitoneal administration of human C9 significantly enhanced survival and also enhanced the protective effect of intraperitoneal human IgG antibodies. The data indicate that C9 deficiency predisposed neonatal rats to invasion by E. coli. The neonatal rat appears to be a suitable model with which to investigate the significance of C9 deficiency.

Age Factors↗
PubMed1983

Immunoaffinity purification of human complement component C9 using monoclonal antibodies.

Five monoclonal antibodies to human C9 were produced. Three of the antibodies had affinities from 1.6 to 3.1 X 10(9) M-1 and 2 were of lower affinity, 0.1 to 0.3 X 10(9) M-1. Four distinct antigenic sites on C9 were detected. The lowest affinity antibody was immobilised on Sepharose 4B and used to isolate C9 from human plasma. A 1200-fold purification was obtained with yield of 30-40%. The purified antigen maintained full cytolytic activity.

Animals↗
PubMed1989

Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies.

Polyclonal rabbit antibodies against melittin recognize human C protein C9 and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence (amino acids 293-296 and 528-532, respectively) that together match a continuous segment in the melittin sequence, i.e., residues 8-16. It has been suggested that the tetrameric and the pentameric regions on C9 form a discontinuous epitope on folded C9 that mimics the structure of melittin. To further test this hypothesis, antibodies to C9-sequence-specific peptides were prepared. Peptides containing either the homologous tetrameric or the homologous pentameric sequence together with short stretches of the respective amino- and carboxyl-terminal flanking regions were synthesized, as well as a composite peptide predicted to resemble the discontinuous epitope as a linear, nine-amino acid sequence. Direct and competitive binding assays demonstrated that the tetrameric and the pentameric sequences are part of the epitope on human C9 that is recognized by anti-melittin IgG. However, only antibodies directed against the complete epitope are capable of inhibiting hemolysis. Because neither anti-tetramer nor anti-pentamer antibodies affect hemolysis whereas anti-melittin and anti-composite antibodies do, we propose that human C9 changes conformation around a hinge located between residues 296 and 528 and that the latter two antibodies inhibit unfolding required for membrane insertion and subsequent hemolysis.

Amino Acid Sequence↗
PubMed1993

The expression of hemolytically active human complement protein C9 in mammalian, insect, and yeast cells.

The cDNA sequence encoding mature human C9 protein and its signal peptide was cloned into three expression vectors for expression in COS-7 (mammalian), Spodoptera frugiperda IPLB-SF-21AE (insect), and Saccharomyces cerevisiae (yeast) cells. In addition, C9 cDNA encoding only the mature protein was fused to the yeast invertase leader sequence (SUC2) and cloned for expression in yeast. Under optimal conditions COS-7 and IPLB-SF-21AE cells secreted recombinant C9 (rC9) at concentrations of about 111 and 700 ng C9/ml culture supernatant, respectively. By comparison S. cerevisiae, whether transformed with C9 cDNA containing its native or yeast invertase leader sequence, secreted only very small amounts of rC9 (5-10 ng/ml). However, upon lysis concentrations of up to 500 ng/mg dry wt were found in yeast cells transformed with C9 cDNA. SDS-PAGE followed by Western blot analysis revealed COS-7 cell and S. cerevisiae expressed rC9 to have a MW similar to that of native C9 purified from human serum, while rC9 from IPLB-SF-21AE cells was about 4 kDa smaller. No hemolytic activity of S. cerevisiae secreted rC9 could be detected and the specific hemolytic activity of S. cerevisiae intracellular rC9 was also very low. However, the specific hemolytic activities of COS-7 and IPLB-SF-21AE secreted rC9 were indistinguishable from that of purified native human C9. Thus, for future studies on the structure and function of C9 where the production of large quantities of mutant protein would be desirable, the baculovirus-insect cell expression system appears to offer considerable advantages.

Animals↗
PubMedDate not supplied

Horse complement protein C9: primary structure and cytotoxic activity.

Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.

Amino Acid Sequence↗
PubMed1990

Quantification of non-activated (native) complement component C9 synthesized by alveolar macrophages from patients with sarcoidosis.

Alveolar macrophages (AM) from sarcoidosis patients synthesize the functional alternative and terminal pathways of complement, and increased complement production may be one of multiple factors in the pathogenesis of sarcoidosis. We thus examined whether AM from sarcoidosis patients produced quantitatively more C9 in vitro than AM from healthy controls. AM from 16 patients with active sarcoidosis and seven healthy controls were cultured under serum-free conditions for 6, 12, 24, 48, or 72 h. A quantitative production of C9 was found in the harvested medium in 10 of 16 sarcoidosis patients. There were no detectable levels of C9 in the seven controls. Activated C9 was found in all patients and in the majority of the controls. C9 was quantified by an enzyme immunoassay based on a monoclonal antibody (M1) to non-activated C9. Our results indicate greater production of C9 by sarcoidosis AM than by their healthy counterparts.

Adult↗
PubMed1997

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes.

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6-7 kb from C9 in a head-to-head or 5' to 5' orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Complement components and terminal complement complex in oesophageal smooth muscle of patients with achalasia.

This study investigates whether patients with achalasia exhibit autoimmune reactions with subsequent complement activation within oesophageal smooth muscle, vessels and neurones. Oesophageal muscular biopsies from 8 patients undergoing surgery for achalasia and from 6 patients operated for oesophageal cancer were investigated by immunofluorescence for the presence of the complement components C1q, C4, C3c, C3d, C9 and the C9 neoantigen of the terminal C5b-C9 complement complex. Tissues were also investigated for the expression of immunoglobulins (G,A,M) and of the antigens of rubella and varicella zoster viruses. In addition, sera of both patient groups were tested for the presence of autoantibodies against Auerbach's plexus. The terminal complement complex C5b-C9 was found within muscle cells from all patients with achalasia but in only one specimen from a patient with cancer. Two patients with achalasia also exhibited the terminal complement complex as well as IgM within ganglion cells. Muscle cells stained positive for the complement component C9 in all five patients with achalasia in whom this test was performed but in none of the control tissues. In addition, sera from four patients with achalasia contained antibodies against Auerbach's plexus. Studies for the complement components C1q, C4, C3c and for antigens of rubella and varicella zoster viruses revealed negative results in all patients and controls. The results of this study suggest that a complement activation is involved in the autoimmune pathogenesis of achalasia. However, the triggering mechanism of this phenomenon remains to be determined.

Adolescent↗
PubMed1984

Muscle fiber degeneration and necrosis in muscular dystrophy and other muscle diseases: cytochemical and immunocytochemical data.

Serial consecutive frozen sections from 164 muscle biopsy specimens were studied cytochemically. We localized calcium and albumin as endogenous markers of extracellular fluid penetration and C3 and C9 complement components as markers of muscle fiber necrosis. In both Duchenne dystrophy and congenital muscular dystrophy, a significant percentage of fibers were overloaded with calcium and penetrated by albumin. Most of these fibers appeared opaque with trichrome stain. C3 and C9 complement components appeared only in necrotic fibers, which invariably were also penetrated by albumin. These observations support previous findings that muscle fiber necrosis is linked to massive inflow of extracellular fluid and complement activation. In addition, in both Duchenne and congenital dystrophies, numerous nonnecrotic fibers are penetrated by calcium-rich extracellular fluid.

Albumins↗
PubMed1988

Comparison between complement and melittin hemolysis: anti-melittin antibodies inhibit complement lysis.

A comparison is made between the hemolytic actions of melittin and the ninth component of complement (C9). Melittin and C9 produce "pores" of similar effective radius in erythrocytes under standardized conditions, and their hemolytic action is suppressed by metal ions at similar concentrations, suggesting a common mechanism. Polyclonal anti-melittin immunoglobulin G (IgG) produced in rabbits retards hemolysis mediated by human C9 in a specific manner. Such antibodies react in several immunoassays with human and monkey C9 but not with C9 from lower animals, and no inhibition of lysis mediated by C9 molecules from these animals is observed. Thus, it is unlikely that anti-melittin IgG reacts with a structural element, such as an amphipathic helix, on human C9 since such structures are also predicted to exist in other C9 molecules. Human C9 and melittin block cross-reactivity in a dose-dependent manner, and anti-melittin IgG recognizes an epitope located between amino acid residues 245 and 390 of human C9 on "Western" blots. Comparison of the melittin and human C9 sequences indicates two regions of complete homology, a tetrapeptide at positions 292-295, and a pentapeptide at positions 527-531 in human C9, corresponding to residues 8-16 in melittin. Inhibition of hemolysis is not caused by blocking of C9 binding to the C5b-8 complex; rather the antibody must dissociate from the bound C9 before lysis ensues, indicating that it interferes with a postbinding event. It is proposed that anti-melittin binds to a conformational epitope on native, folded human C9 and thereby retards unfolding of the molecule, which is required for membrane insertion and hemolysis.

Antibodies↗
PubMed1998

Expression of complement C4 and C9 genes by human astrocytes.

Evidence exists that complement activation is involved in the pathogenesis of Alzheimer's disease (AD). It has been previously demonstrated that central nervous system (CNS) resident cells can synthesize complement proteins. Two key proteins in the complement pathway are the complement C4 and C9 proteins. Using reverse transcription-polymerase chain reaction, ELISA, immunocytochemical and immunoblot techniques, we showed that primary human astrocytes constitutively expressed complement C4 mRNA and protein, and that this was increased when cells were treated with interferon-gamma, but inhibited when cells were treated with interleukin-1beta (IL-1beta). C4 immunoreactivity could be localized to GFAP-positive astrocytes when protein secretion was inhibited. These results indicated that astrocytes could be a source of complement C4 in the human CNS. In addition it was shown that stimulated astrocytes could also express complement C9 mRNA, though C9 protein was not detectable in culture supernatants.

Astrocytes↗
PubMed1997

Response of glial cells and activation of complement following motorneuron degeneration induced by toxic ricin.

Motor nerve transection in adult rats induce a series of metabolic and structural changes in the injured neurons as well as in surrounding glial cells; however, without substantial neuronal degeneration. In the present study we found, in contrast with axotomy, a massive neuronal death in the ipsilateral hypoglossal nucleus following injection of toxic ricin (RCA) into the hypoglossal nerve, which is in line with previous observations. Injection of RCA enables examination of the glial reaction in a situation where neuronal degeneration is profound, which has been the approach in the present study. We found an increase in OX42-, GFAP-, and transferrin-immunoreactivity in microglial, astroglial, and oligodendroglial cells respectively, in the ipsilateral hypoglossal nucleus three to seven days following injection of toxic ricin in the hypoglossal nerve. Proliferation was found in astrocytes as well as in microglial cells, as shown by uptake of bromodeoxyuridine. In addition, the complement cascade was activated locally in the ipsilateral hypoglossal nucleus, as demonstrated by immunohistochemical detection of complement components C3d and C9. Complement activation may serve several effects in the glial-neuronal interactions. Stimulation of phagocytosis by reactive microglia is probably the most important one. Furthermore, the degenerative neuronal somata showed increased immunoreactivity for clusterin, which is a known complement inhibitor, but a decrease in clusterin-mRNA. In conclusion, the glial cell response was in several aspects principally different following massive motorneuron degeneration induced by toxic ricin in comparison to previous findings reported after axotomy.

Animals↗
PubMed1999

Molecular epidemiology of C9 deficiency heterozygotes with an Arg95Stop mutation of the C9 gene in Japan.

Deficiency of the ninth component of human complement (C9) is the most common complement deficiency in Japan, with an incidence of approximately one homozygote in 1000, but is very rare in other countries. Genetic analyses of Japanese C9 deficiency have shown that a C-to-T transition leading to TGA stop codon for Arg95 in exon 4 of the C9 gene (Arg95Stop) is common in Japanese C9 deficiency. To determine the prevalence of heterozygous carriers of the Arg95Stop mutation in a Japanese population, we collected DNA samples from 300 individuals in two of the four main islands of Japan. Heterozygote detection was performed with an allele-specific polymerase chain reaction (PCR) system designed to detect exclusively only one of the normal and mutant alleles, followed by confirmation with PCR/single-strand conformation polymorphism (SSCP) analysis and direct sequencing. Twenty individuals were heterozygous for the Arg95Stop mutation. None was homozygous. The prevalence of carriers of the Arg95Stop mutation was 6.7% (20/300). An estimated frequency (0.12%) of complete C9 deficiency due to homozygous Arg95Stop mutation was consistent with frequencies determined by serological studies.

Arginine↗
PubMed1985

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 cytolytically 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 3B1, 3D8, and 2F3), sheep (clones 3D8 and 2F3) and guinea-pig (clone 3D8) neoantigens were identified. Three of four tested clones (3D8, 2F3, 1A12) 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.

Animals↗
PubMed1984

Circular polymerization of the membranolytic ninth component of complement. Dependence on metal ions.

The ninth component of complement (C9) polymerizes to tubules either spontaneously at a very slow rate (3 days at 37 degrees C) or within minutes if induced by C5b-8 as part of the membrane attack assembly (Tschopp, J., Podack, E. R., and Müller-Eberhard, H. J. (1984) J. Immunol., in press). The spontaneous formation of tubular sodium dodecyl sulfate-resistant C9 polymers was completely inhibited in the presence of 1 mM EDTA. In contrast, the presence of Cu2+, Zn2+, and Cd2+ accelerated C9 polymerization. Zn2+ was the most efficient metal ion. At a molar ratio of one Zn2+ per monomeric C9, all C9 polymerized within 2 h. At Zn2+ concentrations above 10(-3) M, tubular C9 formation was inhibited due to nonspecific aggregation of C9. It is concluded that the presence of metal ions is required for the formation of tubular poly C9 and that Zn2+ in particular catalyzes C9 polymerization.

Cadmium↗
PubMed1990

Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Affinity Labels↗