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PubMed1991

Resistance of Escherichia coli to osmotically introduced complement component C9.

Investigation into the action of osmotically introduced C9 in Escherichia coli (in the absence of any other complement components) revealed that C9 could inhibit inner membrane respiration and cause a decrease in the viability of cells that were normally complement sensitive. This effect is analogous to the loss of inner membrane function and viability due to the assembly of the C5b-9 complex on these cells. Complement-resistant cells showed no such inhibition of respiration or loss of viability when subjected to the osmotic introduction of C9. The reason for this failure of C9 to affect complement-resistant cells was explored to determine whether this resistance to C9 was due to an inability of proteins in general to be osmotically introduced into the complement-resistant cells. The protein toxins melittin and colicin E1 were showed to be able to kill these complement-resistant cells (as well as complement-sensitive cells) when osmotically introduced into the periplasm. Therefore, cellular resistance to osmotically introduced C9 is not due to an inability of proteins to be introduced into the cells and may be related to a mechanism of cellular resistance to the C5b-9 complex.

Bacterial Outer Membrane Proteins↗
PubMed1985

Proteolytic modification of human complement protein C9: loss of poly(C9) and circular lesion formation without impairment of function.

We have compared the ability of thrombin-cleaved C9 (C9n) with that of native C9 to produce tubular or ring-like poly(C9) and to express the classical complement lesion on target membranes. Three procedures were used to produce poly(C9): (i) limited proteolysis with trypsin, (ii) interaction with small unilamellar lipid vesicles, and (iii) incubation with a 2- to 4-fold molar excess of ZnCl2. In contrast to C9, which could be converted to tubular poly(C9), C9n was converted to smaller peptides by the first procedure and was aggregated into string-like poly(C9) by the other two methods. C9-depleted human serum (R-9 serum) was reconstituted with either C9 or C9n and these sera were then used to lyse sensitized sheep erythrocytes. Numerous classical complement lesions could be detected on ghost membranes obtained from cells lysed by C9-reconstituted R-9 serum but only a few on ghost membranes produced by C9n-reconstituted R-9 serum. C9n was shown to be hemolytically as active as C9 even when tested under "single-hit" conditions and it was about twice as efficient when compared with C9 in releasing sucrose and inulin from resealed ghosts. These results are interpreted to indicate that formation of the classical complement lesion is only incidental to lysis and not an obligatory event and that enlargement of the "functional pore size" of the complement lesion is not linked to formation of a circular membrane attack complex.

Complement C9↗
PubMed1988

Detecting distant homologies of mosaic proteins. Analysis of the sequences of thrombomodulin, thrombospondin complement components C9, C8 alpha and C8 beta, vitronectin and plasma cell membrane glycoprotein PC-1.

Recognition of homologies may give hints about the structure and function of proteins; therefore, we are developing strategies to aid sequence comparisons. Detecting homology of mosaic proteins is especially difficult since the modules constituting these proteins are usually distantly related and their homology is not readily recognized by conventional computer programs. In the present work we show that the rules of the evolution of mosaic proteins can guide the identification of modules of mosaic proteins and can delineate the group of sequences in which the presence of homologous sequences may be expected. By this approach we can concentrate the search for homology to a limited group of sequences; thus ensuring a more intense and more fruitful search. The power of this approach is illustrated by the fact that it could detect homologies not identified by earlier methods of sequence comparison. In this paper we show that thrombomodulin contains a domain homologous with animal lectins, that complement components C9, C8 alpha and C8 beta have modules homologous with one of the repeat units of thrombospondin and that the somatomedin B module of vitronectin is homologous with the internal repeats of plasma cell membrane glycoprotein PC-1.

Amino Acid Sequence↗
PubMedDate not supplied

Domain structure, functional activity, and polymerization of trout complement protein C9.

The 3' region of trout C9 has been resequenced and found to differ from the previously published sequence (Stanley and Herz, EMBO J. 6:1951; 1987). In contrast to other sequenced C9 molecules, but in common with the other terminal complement components, trout C9 was found to contain an additional carboxy terminal thrombospondin domain. This domain does not restrict polymerization, as has been previously suggested (Stanley and Luzio, Nature 334:475; 1988), since alternative pathway activation of trout complement by rabbit erythrocytes lead to the formation of circular membrane attack complement lesions on the erythrocyte membrane. Although the trout C9 molecule is larger than human C9, the diameters of circular trout membrane attack complexes were approximately 30% smaller than their human counterparts. No lysis of erythrocytes bearing human C5b-7 or C5b-8 complexes was detected following incubation with trout serum containing EDTA, which suggests that trout C8 and C9 are unable to bind to human C7 and C8, respectively. Finally, trout and human serum were equally effective at killing the human serum-sensitive strain Salmonella minnesota Re595.

Amino Acid Sequence↗
PubMed2002

The administration of complement component C9 augments post-ischemic cerebral infarction volume in neonatal rats.

To determine whether ischemic cerebral infarction is mediated in part by complement component C9, C9-deficient neonatal rats were subjected to unilateral cerebral ischemia. Brains were harvested 24 h later, stained with 2,3,5-triphenyl tetrazolium chloride, and cerebral infarct volumes were quantified by computer-based planimetry. Compared with buffer, prophylactic intraperitoneal (i.p.) administration of the complement inhibitors soluble complement receptor type 1 (sCR1), a molecular hybrid of sCR1 and the selectin inhibitor sialyl Lewis x (sCR1-sLex), or cobra venom factor did not affect the cerebral infarct volume. In contrast, i.p. human C9 (75 microg/g body weight) significantly increased the volume of infarct located 6 through 10 mm posterior to the frontal pole. Therefore, in the post-ischemic brain, C9 was neurotoxic and augmented the focal cerebral infarct volume.

Animals↗
PubMed1994

Supplemental complement component C9 enhances the capacity of neonatal serum to kill multiple isolates of pathogenic Escherichia coli.

Previous studies demonstrated that, compared with adult serum, neonatal serum contained a diminished concentration of complement component C9 and that supplemental C9 enhanced the capacity of neonatal serum to kill an isolate of Escherichia coli. Therefore, experiments were designed to determine the mechanisms by which supplemental C9 enhances the bactericidal capacity of neonatal serum and to determine whether supplemental C9 enhances the capacity of neonatal serum to kill several different pathogenic strains of E. coli. A radiobinding assay and immunogold electron microscopy using a monoclonal anti-C9 antibody revealed that, compared with 40% adult serum, neonatal serum deposited a diminished quantity of C9 onto E. coli O7w:K1:NM. Supplemental C9 (75 mg/L) significantly enhanced the quantity of C9 deposited by the neonatal serum. Treatment with 10 mM MgEGTA (a mixture of 100 mM MgCl2 and 100 mM EGTA that blocks activation of the classic complement pathway but leaves the alternative pathway intact) abolished the capacity of neonatal serum to deposit C9 and to kill the bacteria. Supplemental C9 enhanced the capacity of neonatal serum to kill eight different blood isolates of E. coli. Therefore, supplemental C9 enhanced the capacity of neonatal serum to kill E. coli by increasing the total quantity of C9 deposited via activation of the classic complement pathway. Neonatal serum contained sufficient quantities of classic pathway components, other than C9, to deposit the supplemental C9 onto E. coli and to enhance bacterial killing. The bactericidal activity of neonatal serum against multiple isolates of pathogenic E. coli was increased after C9 supplementation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1983

Mechanisms for the spontaneous formation of covalently linked polymers of the terminal membranolytic complement protein (C9).

Purified human C9 spontaneously polymerizes upon prolonged incubation at 37 degrees C, and a fraction of these C9 polymers becomes resistant to dissociation by sodium dodecyl sulfate (SDS) and reducing agents. We examined possible mechanisms for this spontaneous covalent linking of C9. The following results are consistent with the conclusion that the formation of the covalently linked C9 polymer involves disulfide linking. 1) In addition to the SDS/dithiothreitol (DTT)-resistant C9 polymer (Mr = 950,000), disulfide-linked C9 dimers and trimers were formed upon incubation of C9 at 37 degrees C for 64 h. 2) The C9 polymer formed upon incubation at 37 degrees C for 64 h was resistant to dissociation by 6 M guanidine hydrochloride, 20 mM DTT but was dissociated by 6 M guanidine thiocyanate alone, yielding disulfide-linked C9 oligomers. 3) The formation of the SDS/DTT-resistant C9 polymer was completely inhibited by 1 mM iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), while DTNB enhanced the formation of disulfide-linked C9 oligomers. 4) A significant amount of free sulfhydryl group was detected in the polymerized C9 samples with various SH-specific reagents, though native C9 reacted with none of these reagents. In addition, inhibition by 1 mM iodoacetamide of C9 disulfide linking inhibited the self-association of C9 as analyzed by gel filtration on TSK-G4000 SW, whereas enhancement by 1mM DTNB of C9 disulfide linking enhanced C9 self-association. Thus, these results indicate that C9 disulfide linking that occurs upon C9 polymerization is an intrinsic property of C9 which is of importance in the formation of the stable C9 polymer structure.

Complement C9↗
PubMed2003

An abnormal but functionally active complement component C9 protein found in an Irish family with subtotal C9 deficiency.

Two independently segregating C9 genetic defects have previously been reported in two siblings in an Irish family with subtotal C9 deficiency. One defect would lead to an abnormal C9 protein, with replacement of a cysteine by a glycine (C98G). The second defect is a premature stop codon at amino acid 406 which would lead to a truncated C9. However, at least one of two abnormal proteins was present in the circulation of the proband at 0.2% of normal C9 concentration. In this study, the abnormal protein was shown to have a molecular weight approximately equal to that of normal C9, and to carry the binding site for monoclonal antibody (mAb) Mc42 which is known to react with an epitope at amino acid positions 412-426, distal to 406. Therefore, the subtotal C9 protein carries the C98G defect. The protein was incorporated into the terminal complement complex, and was active in haemolytic, bactericidal and lipopolysaccharide release assays. A quantitative haemolytic assay indicated even slightly greater haemolytic efficiency than normal C9. Epitope mapping with six antihuman C9 mAbs showed the abnormal protein to react to these antibodies in the same way as normal C9. However, none of these mAbs have epitopes within the lipoprotein receptor A module, where the C98G defect is located. The role of this region in C9 functionality is still unclear. In conclusion, we have shown that the lack of a cysteine led to the production of a protein present in the circulation at very much reduced levels, but which was fully functionally active.

Aged↗
PubMed1987

Bacterial killing by complement. C9-mediated killing in the absence of C5b-8.

The ability of serum complement to kill Gram-negative bacteria requires assembly of the membrane attack complex (MAC) on the cell surface. The molecular events that lead to cell killing after MAC assembly are unknown. We have investigated the effect of C9 on bacterial survival in the presence and absence of its receptor, the C5b-8 complex, on the outer membrane. A fluorescence assay of the membrane potential across the inner bacterial membrane revealed that addition of C9 to cells bearing the performed C5b-8 complex caused a rapid and complete dissipation of the membrane potential. No fluorescence change was observed in serum-resistant strains of Escherichia coli. Addition of trypsin, after C9 was bound to C5b-8, did not rescue the cells from the lethal effects of C9. Furthermore, assays of cell killing kinetics and C9 binding indicate that formation of tubular poly(C9) is not required for killing. When C9 was introduced into the periplasmic space in the absence of its receptor by means of an osmotic shock procedure, cell killing occurred. Other proteins, such as C8 or serum albumin, were not toxic, and C9 was ineffective against two resistant strains. The results presented here and previously [Dankert & Esser (1986) Biochemistry 25, 1094-1100], when considered together, indicate that the 'lethal unit' in complement killing of some Gram-negative bacteria is a C9-derived product that acts by dissipation of cellular energy.

Blood Bactericidal Activity↗
PubMed1989

Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9.

The nucleotide sequence coding for the cytotoxic T-lymphocyte (CTL) protein perforin 1 (P1) has been determined and the corresponding protein sequence has been derived. Murine CTL cDNA libraries contained in the vector lambda gt11 were screened by using a monospecific antiserum to purified P1. Three recombinant phages were isolated and their cDNA inserts were sequenced. The derived protein sequence contains 554 amino acids and displays, as expected, considerable homology with certain functional domains in the complement components C9, C8 alpha, C8 beta, and C7. The identity of P1 cDNA clones was verified by prokaryotic expression and the reactivities of antisera produced to the expressed proteins. In addition, antisera were produced to two synthetic peptides located in the center and C-terminal portions of P1. All antisera reacted with purified P1. In Northern blot analyses, P1 cDNA probes recognized a 2.9-kilobase mRNA only in CTL. Perforin mRNA was found in all cloned CTL and in all mixed lymphocyte reactions that gave rise to cytotoxic cells. Perforin mRNA was also detected in virus-specific CTL that had been generated in vivo and isolated from liver tissue of mice infected with lymphocytic choriomeningitis virus. The cell-specific expression of perforin is consistent with its postulated role in cytolysis.

Amino Acid Sequence↗
PubMed1985

Multimeric complement component C9 is necessary for killing of Escherichia coli J5 by terminal attack complex C5b-9.

We studied the molecular composition of the complement C5b-9 complex required for optimal killing of Escherichia coli strain J5. J5 cells were incubated in 3.3%, 6.6%, or 10.0% C8-deficient serum previously absorbed to remove specific antibody and lysozyme. This resulted in the stable deposition after washing of 310, 560, and 890 C5b67 molecules per colony-forming unit, respectively, as determined by binding of 125I-labeled C7. Organisms were then incubated with excess C8 and various amounts of 131I-labeled C9. Plots of the logarithm (base 10) of E. coli J5 cells killed (log kill) vs. C9 input were sigmoidal, confirming the multihit nature of the lethal process. When C9 was supplied in excess, 3300, 5700, and 9600 molecules of C9 were bound per organism for cells bearing 310, 560, and 890 C5b-8 complexes, respectively, leading to C9-to-C7 ratios of 11.0:1, 10.8:1, and 11.4:1 and to log kill values of 1.3, 2.1, and 3.9. However, at low inputs of C9 that lead to C9-to-C7 ratios of less than 3.3:1, no killing occurred, and this was independent of the number of C5b-9 complexes bound. Formation of multimeric C9 at C9-to-C7 ratios permissive for killing was confirmed by electron microscopy and by binding of 125I-labeled antibody with specificity for multimeric but not monomeric C9. These experiments are the first to demonstrate a biological function for C9 polymerization and suggest that multimeric C9 is necessary for optimal killing of E. coli J5 cells by C5b-9.

Cell Membrane↗
PubMed1995

The kinetics and distribution of C9 and SC5b-9 in vivo: effects of complement activation.

Many diseases associated with complement activation are characterized by tissue deposition of components of the terminal complement complex (TCC). The ninth component of complement (C9) plays an important role in the cytolytic effects, and may contribute to the non-lethal cell-regulating functions of the TCC. In this study we examined the behaviour of radiolabelled human C9 and its soluble complexed form SC5b-9 in vivo in order to determine the effects of complement activation on its turnover, distribution and molecular size. In normal rabbits the metabolic parameters of 125I-C9 (median and range) were: plasma half-life (t1/2) 25.9 (20.6-29.5) h, fractional catabolic rate (FCR) 5.7 (5.3-7.0)%/h, and extravascular/intravascular ratio (EV/IV) 0.7 (0.6-1.1). The distribution of radiolabelled C9 amongst body tissues was similar to that observed for rabbit serum albumin (RSA). Activation of the complement cascade with i.v. injection of cobra venom factor (CVF) resulted in rapid disappearance of C9 from the plasma and accumulation of protein-bound radiolabeled in the spleen (exceeding the plasma concentration) and the liver. RSA metabolism and distribution were unaffected by CVF. Fine performance liquid chromatography (FPLC) gel filtration of plasma samples suggested that monomeric C9 was the only major radiolabelled protein present during normal turnovers, whereas CVF administration was accompanied by the prompt appearance of a high mol. wt species consistent in size with SC5b-9. When injected directly, 125I-SC5b-9 disappeared rapidly from the plasma, falling by 50% in 0.7 (0.6-0.8) h, and less than 15% remaining after 4 h with accumulation of protein-bound label in the spleen and liver. These results demonstrate the complexity of C9 metabolism during complement activation.

Animals↗
PubMed1987

Topological mapping of complement component C9 by recombinant DNA techniques suggests a novel mechanism for its insertion into target membranes.

cDNA molecules coding for mouse and trout C9 have been isolated and the derived amino acid sequences compared with that of human C9. Regions of high homology between the closely related species (mouse and human) correlate with putative domains in the protein structure supporting a model of C9 having five globular domains. Comparison between the more distant species (trout and human) suggests regions of particular importance to C9 structure and function. In addition the three related sequences allow the secondary structure to be predicted with more confidence and we have tested the prediction by mapping surface features of the protein. Reported here is a recombinant DNA approach to fine mapping of antibody epitopes. Two of the putative domains of C9 are connected by a stretch of about 40 amino acid residues in which features characteristic of individual conformational forms of C9 are concentrated. We suggest that this region might act as a hinge allowing the rearrangement of globular domains necessary for membrane insertion. In the membrane inserting domain one highly conserved sequence has the potential to form an amphipathic alpha-helix once it is buried in the lipid bilayer. These features suggest a novel mechanism for the irreversible, post-translational insertion of C9 into target membranes.

Amino Acid Sequence↗
PubMed1994

A synthetic peptide from complement protein C9 binds to CD59 and enhances lysis of human erythrocytes by C5b-9.

The membrane glycoprotein CD59 protects host cells from homologous complement attack by inhibiting the assembly of the membrane attack complex. CD59 binds to C8 and C9 in the nascent membrane attack complex and interferes with C9 membrane insertion and polymerization. We show here that a synthetic peptide from the putative C9 hinge region, postulated to be involved in the rearrangement of C9 globular domains during membrane insertion, binds specifically to CD59 and enhances lysis of human erythrocytes by the terminal complement C5b-9 complex. The peptide, C9H, caused a dose-dependent increase in the sensitivity of human erythrocytes to C5b-9-mediated lysis by interfering with the final C9 binding and/or membrane insertion step. C9H exhibited species-specificity, since it had no activity against guinea pig C8 and C9 or on the putative functional homologues of CD59 in guinea pig erythrocytes. A direct association between CD59 and C9H was suggested by two different binding experiments: C9H inhibited the binding of 125I-labeled CD59 to immobilized C9, and C9H immobilized to microtiter plates bound purified CD59 and selectively recognized CD59 from extracts of detergent-solubilized human erythrocyte membranes. These data indicate that the peptide C9H corresponds to a region of the CD59 binding site of C9.

Amino Acid Sequence↗
PubMed1989

A biotin-avidin sandwich ELISA for quantification of intact complement component C9. The sera from hereditary C9 deficient individuals completely lack C9.

A two-site sandwich ELISA method was developed for quantitating intact C9 protein using MoAb P40 (anti-C9b antibody). This antibody reacted with monomeric C9 but not with polymerized C9. MoAb P40 was used as a capture antibody and MoAb X195 (anti-C9a antibody) as a detection antibody. This method is highly sensitive and can detect approximately 0.5 ng/ml of native C9. No cross-reactivities of either C6, C7, or C8 were observed even at concentrations of 10 micrograms/ml per component. In addition, this method allows for measurement of only intact C9 molecules, eliminating the interference of polymerized C9 or inactivated C9. Using this assay, no C9 at all was detected in sera from inherited C9 deficient individuals, including both healthy blood donors and patients with meningococcal meningitis; although by hemolytic assay, C9 levels were reported to be less than 0.2% those of NHS. Therefore, this two-site sandwich ELISA method can replace the hemolytic assay, and is especially useful for measuring small amounts of C9 in serum.

Animals↗