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PubMed1984

Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion.

Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.

Animals↗

PubMed1989

N-deglycosylation of human complement component C9 reduces its hemolytic activity.

The effect of enzymatic deglycosylation of human complement component C9 on its hemolytic activity was investigated. Treatment of native C9 (Mr 71,000) with glyocpeptidase F (PNGase F) results in a stepwise decrease of the mol. wt. The formation of an Mr 67,000 peptide which is further converted to Mr 63,000 suggests that there are two N-linked carbohydrate chains per C9 polypeptide. Removal of approximately 88% of the N-linked oligosaccharides results in 80% reduction of the hemolytic activity (CH50). The completely N-deglycosylated Mr 63,000 peptide contains a remaining amount of 25% of the total carbohydrates of native C9. These glycans are assumed to be O-linked and predominantly attached to the C9a part of C9. The electrophoretic mobility of C9 is not affected by endoglycosidase F or H treatments revealing that the two N-linked glycans are of the tri- or tetra-antennary complex type. Cleavage of terminal sialic acids from native C9 by neuraminidase results in an Mr 67,000 product with nearly unaltered hemolytic activity. In contrast to other glycoproteins in which deglycosylation remained without major effects on their functional activity, our findings suggest that the N-linked carbohydrates are required for full expression of hemolytic activity of C9.

Amidohydrolases↗

PubMed1984

Immunolocalization of complement component C9 on necrotic and non-necrotic muscle fibres in myositis using monoclonal antibodies: a primary role of complement in autoimmune cell damage.

Monoclonal antibodies to human complement component C9 were used to localise the membrane attack complex (MAC) of human complement on muscle fibres from patients with autoimmune myositis. A pigeon erythrocyte 'model' system was used to screen the available monoclonal antibodies for their ability to bind C9 after its insertion into the MAC. Three of the five antibodies tested were demonstrated, using radioiodine- or rhodamine-labelled second antibody, to bind. The antibody giving maximum binding in this system was used to demonstrate that MACs were present on histologically normal as well as on necrotic fibres in myositis. No binding was seen in muscle from control subjects. These results suggest a primary role of complement in the pathogenesis of muscle fibre necrosis in myositis.

Adult↗

PubMed1988

Human complement protein C9 is a calcium binding protein. Structural and functional implications.

Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca2+/mol of C9 with a dissociation constant of 3 micron as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element or domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although we do not know yet the precise structural and functional role of the bound calcium, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.

Calcium↗

PubMed1979

Isolation of late complement components by affinity chromatography: I. Purification of the human complement component C9 and production of a C9-defective human serum.

A new procedure for the isolation of the human complement component C9 is described. This procedure offers the possibility to prepare functionally pure C9 in a one-step procedure with a high recovery of 10-22% of the biological activity. The 125iodinated C9 had a molecular weight of 78,000 daltons and was only contaminated in traces with other proteins. Further purification by absorption with an "anti-impurity" column lead to a C9 preparation which behaved as a homogenous single polypeptide chain in SDS polyacrylamide gel electrophoresis after reduction with mercaptoethanol. It formed a single bell-shaped precipitate in crossed immunoelectrophoresis with antibodies against human serum in the gel of the second dimension. The recovery of the biological activity after the second purification step was in the order of 6-10%. Both preparative steps could be performed within a few hours 450 microgram C9 protein were isolated from 135 ml human serum. A human serum completely defective in C9 was prepared by the extensive absorption of a smaller volume of human serum proteins with the anti-C9 column.

Complement C9↗

PubMed1997

Mutation of recombinant complement component C9 reveals the significance of the N-terminal region for polymerization.

Complement component C9 binds to C5b-8 sites on target cells and polymerizes to form the membrane attack complex (MAC). The aim of the work reported here was to discover which region within C9 was responsible for protecting the globular protein against self-polymerization. Computer prediction modelling highlighted the domain at the N-terminus of C9, which was then investigated by site-directed mutagenesis. The mutated proteins were expressed using insect cells infected with baculovirus. Removal of 16, 20 or 23 amino acids at the N-terminus of C9 resulted in inactivation due to self-polymerization. In contrast, removal of 4, 8 or 12 amino acids resulted in a C9 that did not polymerize spontaneously, had two to threefold enhanced lytic activity on erythrocytes, and had increased binding to C5b-8 sites on rat neutrophils. These results suggest that the domain within the first 16 amino acids at the N-terminus of C9 is crucial in preventing the self-polymerization of the globular protein. We have also found that C9 contains a motif (27WSEWS31) common to a family of cytokine receptors that is similar to a tryptophan-rich motif (WEWWR) of the membrane pore formers, thiol-activated cytolysins. Mutation of this motif in C9 resulted in polymerized protein, consistent with this site keeping the N-terminus in a protected conformation and preventing premature self-polymerization.

Amino Acid Sequence↗

PubMed1991

Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.

The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a membrane surface, and we propose that this facilitates membrane insertion and refolding of the protein into an amphiphilic conformation.

Buffers↗

PubMed1980

Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis.

The terminal and lytic complement component (C9) was localized at the motor end-plate in acquired autoimmune myasthenia gravis (MG) by the immunoperoxidase method, with adequate preservation of fine structure and negligible background staining. C9 was localized on short segments of the postsynaptic membrane on degenerated fragments of the junctional folds shed into the synaptic space, and on disintegrating junctional folds. An inverse relationship was noted between the structural integrity of the junctional folds and the abundance of C9 at a given end-plate region. Destruction of junctional folds by complement may induce relocation of the nerve terminal and increased spatial separation of end-plate regions on the muscle fiber. Destruction of junctional folds by the complement membrane attack complex is a cause of the acetylcholine receptor deficiency at the MG end-plate, but antibody-dependent modulation of the receptor may also contribute to deficiency of the receptor. In certain disorders other than autoimmune MG, pathological mechanisms other than complement-mediated lysis may affect the structural integrity of the postsynaptic region.

Adolescent↗

PubMed1999

The architectural transition of human complement component C9 to poly(C9).

Several regions of C9 including three cysteine-rich modules homologous to those in thrombospondin (TS), the low density lipoprotein receptor (LDL), the epidermal growth factors (EDGF), as well as two middle sections of the polypeptide chain were expressed in bacteria. Antibodies derived from these segments were used to probe the relative exposure of epitopes in C9 and poly(C9) using ELISAs. The results indicated that the TS and LDL modules are fully exposed in both monomer and polymer; however, the middle region of the polypeptide chain is buried in the monomer but external in the polymer. Using specified conditions, Fab fragments to the TS and LDL modules did not block C9 polymerization, but those to the middle region of the polypeptide chain and to some extent to the EDGF module did so. Immuno-electron microscopy of poly(C9) indicated that the C9 polypeptide chain assumes a 'U' shape, in which the TS and LDL modules are located on the upper rim. The EDGF module is located on the lower edge of the upper rim, and midsection of the polypeptide chain constructs the barrel of the tubule. Computer assisted contrast enhancement of select electron micrograph images of poly(C9) allowed the clear visualization of each subunit. These were seen to have a volute shape. The upper rim is composed of whorls that are apparently not in lateral contact. It is concluded that the TS and LDL modules do not participate directly in polymerization but cover the hydrophobic central region of the polypeptide chain in the monomer. As a consequence of circular polymerization the midsection of the polypeptide chain becomes exposed as each C9 lengths to fashion a volute form. reserved.

Complement C9↗

PubMed1989

The gene for human complement component C9 mapped to chromosome 5 by polymerase chain reaction.

The gene for human complement component C9 has been mapped to chromosome 5. This was achieved by using a novel application of the polymerase chain reaction to amplify specifically the human C9 gene on a background of rodent DNA in somatic cell hybrids. The assignment to chromosome 5 was confirmed by in situ hybridization to human metaphase chromosomes, giving a regional localization of 5p13.

Animals↗

PubMed1993

The size, shape and stability of complement component C9.

Electron microscopy of specimens of C9 tilted through 90 degrees visualized this protein to be a globular ellipsoid with dimensions of 77 x 70 x 52 A. To check the congruence of this observation with physical properties of the molecule, hydrodynamic parameters for C9 were determined. From this work a frictional ratio of 1.32 was calculated. C9 was compared with several other proteins of similar frictional ratios whose tertiary structures are known. All examples found of such proteins whose frictional ratios were between 1.26 and 1.37 are either heart-shaped or globular ellipsoids, but none are prolate ellipsoids. By comparison the size and shape of C9 determined by electron microscopy are congruent with its hydrodynamic parameters. Both electron microscopy and physical measurements suggest that the length (110-120 A) of C9 determined by neutron and X-ray scattering experiments is an overestimate. The source of the discrepancy was identified by the demonstration that the high concns of C9 employed in neutron and X-ray scattering work lead to aggregation of the protein. Thus, investigations involving neutron and X-ray scattering were measuring polydisperse solutions of C9. The deduced value of the radius of gyration from that work (33-35 A) is now recognized as being statistical and significantly higher than the correct value of monomeric C9 (26 A), which was calculated from electron microscopy measurements. Also high-resolution electron microscopy clearly visualized poly(C9) to be a barrel-stave construct. These results suggest that monomeric C9 must undergo a major conformational alteration to extend by 55-70 A in order to self-associate laterally in order to fashion the cylindrical poly(C9).

Complement C9↗

PubMed1998

Complement component C9 enhances the capacity of beta-lactam antibiotics to kill Escherichia coli in vitro and in vivo.

Complement component C9 is required for rapid complement-mediated killing of Escherichia coli. In this report, the influence of supplemental C9 on the bactericidal and protective effects of beta-lactam antibiotics in neonates was assessed. By rocket immunoelectrophoresis, the intrinsic C9 concentrations of pooled serum from both human and rat neonates was less than 20% of adult levels. Supplemental C9 purified from human plasma enhanced the capacity of ampicillin-treated serum from human neonates to impair the survival of E coli O7:K1:NM (P < 0.02). Similarly, supplemental C9 enhanced the capacity of cefotaxime-treated neonatal rat serum to impair the survival of E coli O1:K1:NM (P < 0.05). Moreover, the intraperitoneal administration of C9 enhanced the survival of cefotaxime-treated neonatal rats that were septic with E coli (P < 0.05). These observations may contribute to the development of new strategies, such as augmentation of complement component serum concentrations, to reduce the morbidity and mortality of neonatal E coli sepsis.

Adult↗

PubMed1994

Altered glycosylation and selected mutation in recombinant human complement component C9: effects on haemolytic activity.

Recombinant wild-type and mutated forms of human complement component C9 have been synthesized in baculovirus-infected insect cells. Wild-type recombinant C9 was indistinguishable from native C9, as judged by haemolytic activity, trypsin and alpha-thrombin digestion, reaction with antibodies to C9, enzymatic deglycosylation to the same core size and polymerization in the presence of Zn2+. Replacement of the native signal peptide with the honey-bee melittin signal peptide, and replacement of Spodoptera frugiperda (Sf9) cells with Trichoplusia ni cells produced yields of 5 micrograms C9/ml supernatant. Three C9 mutants were generated; one mutant, with four acidic residues changed to alanines in a putative calcium-binding site, had the same biological activity as recombinant C9. Another mutant, lacking 23 N-terminal amino acids, previously showing increased polymerization when produced in vitro, polymerized on secretion, rendering it inactive. It was not possible to demonstrate haemolytic activity of the third mutant, cysteines 33 and 36 mutated to alanine, as it was secreted a hundredfold less than the wild-type protein.

Animals↗

PubMed1991

The relationship between polymerization of complement component C9 and membrane channel formation.

C9 was studied with the objective to clarify the relationship between the process of C9 polymerization and membrane channel formation. Conditions that favor C9 polymerization include low ionic strength and calcium ion in the buffer. Moreover, polymerization is dependent on the concentration of C9. Calcium ion evokes about a threefold increase in the affinity constant for C9 self-association, and at 0 degrees C it imparts reversible amphiphilic properties in the molecule. These were discerned by measuring increases in the degree of reversible nonspecific binding of C9 to hydrophobic (tyramine-zymosan) and hydrophilic (arginyl-glutamyl-zymosan) supports as well as to erythrocytes. At 0 degrees C the hydrophilic-to-amphiphilic alteration of C9 is reversible, but upon incubation at 37 degrees C this transition is rendered permanent with the formation of poly(C9). A functional relationship between C9 polymerization and cytolysis was demonstrated by showing that polymerizing C9 can lyse reduced and alkylated erythrocytes. By studying comparative radiolabeling of tyrosine side chains within thrombin-nicked C9 and its polymerized form, it was demonstrated that upon polymerization the membrane-binding site of C9 becomes exposed. It is concluded that the process of circular polymerization of C9 causes a hydrophilic-to-amphiphilic transition that is required for membrane perforation and channel formation.

Animals↗

PubMed1986

Complement component C9 in Graves' disease.

C9, the terminal component of complement, is the key part of the membrane attack complex formed as a result of complement activation; it has also been reported to be an acute phase protein. Its potential role in Graves' disease has been studied by measuring plasma C9 concentrations using an automated two-site immunoradiometric assay employing monoclonal antibodies, whose binding to thyroid tissue has also been investigated. The plasma C9 concentration in patients with hyperthyroid Graves' disease (86.3 +/- 21.6 mg/l, mean +/- SD; n = 49) was significantly increased (P less than 0.001) compared with normal subjects (60.4 +/- 13.4 mg/l; n = 48). In contrast, the plasma concentration of C-reactive protein, a marker of the acute phase response, was not significantly different between the two groups. The plasma C9 concentration in patients with hyperthyroid Graves' disease decreased significantly (P less than 0.01) after treatment with antithyroid drugs (carbimazole or methimazole; n = 14), but not after radioactive iodine (131I) treatment (n = 18). Immunohistochemical staining demonstrated that monoclonal antibody to C9 bound to the basement membranes of thyroid follicular cells of Graves' thyroid tissue but not to normal thyroid tissue. Radiolabelled monoclonal antibody to C9 bound to membrane fragments prepared from thyroid glands from two patients with Graves' disease. We conclude that C9, and by implication the membrane attack complex, may be involved in the pathogenesis of Graves' disease.

Adult↗

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↗

PubMed1999

Presence of vitronectin and activated complement factor C9 on ventriculoperitoneal shunts and temporary ventricular drainage catheters.

OBJECT: The pathogenesis of cerebrospinal fluid (CSF) shunt infection is characterized by staphylococcal adhesion to the polymeric surface of the shunt catheter. Proteins from the CSF--fibronectin, vitronectin, and fibrinogen--are adsorbed to the surface of the catheter immediately after insertion. These proteins can interfere with the biological systems of the host and mediate staphylococcal adhesion to the surface of the catheter. In the present study, the presence of fibronectin, vitronectin, and fibrinogen on CSF shunts and temporary ventricular drainage catheters is shown. The presence of fragments of fibrinogen is also examined. METHODS: The authors used the following methods: binding radiolabeled antibodies to the catheter surface, immunoblotting of catheter eluates, and scanning force microscopy of immunogold bound to the catheter surface. The immunoblot showed that vitronectin was adsorbed in its native form and that fibronectin was degraded into small fragments. Furthermore, the study demonstrated that the level of vitronectin in CSF increased in patients with an impaired CSF-blood barrier. To study complement activation, an antibody that recognizes the neoepitope of activated complement factor C9 was used. The presence of activated complement factor C9 was shown on both temporary catheters and shunts. CONCLUSIONS: Activation of complement close to the surface of an inserted catheter could contribute to the pathogenesis of CSF shunt infection.

Adsorption↗

PubMed1987

A unique epitope exposed in native complement component C9 and hidden in the terminal SC5b-9 complex enables selective detection and quantification of non-activated C9.

Recently, monoclonal antibodies recognizing epitopes exposed in activation products of complement but hidden in the native components have been characterized and used for selective quantification of the activated protein. We now demonstrate that an epitope in the native component C9 is hidden in the terminal SC5b-9 complex. A monoclonal antibody against this epitope enabled selective detection of C9 without influence of the amount of SC5b-9 present. This antibody recognizing native soluble C9 was used to construct a quantitative double-antibody ELISA with unique sensitivity and specificity. Combination of this assay with an assay previously described for selective quantification of the SC5b-9 complex provides an important tool for evaluating terminal pathway activation of complement.

Animals↗