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PubMed1993

Interactions of soluble CD59 with the terminal complement complexes. CD59 and C9 compete for a nascent epitope on C8.

Human CD59-Ag is a glycophosphoinositol lipid-anchored inhibitor of the membrane attack complex of complement (MAC). We have examined the interactions of CD59 with the terminal complement complexes using soluble CD59 purified from human urine (CD59U). CD59U bound to SC5b-8, SC5b-9, and MAC complexes when present during their formation. When SC5b-8, SC5b-9, and MAC were allowed to perform, progressively less 125I-CD59U bound to the complexes. Terminal SC5b-9 complexes isolated from activated sera no longer bound CD59U, indicating that the binding sites had become inaccessible in the fully assembled SC5b-9 complex. Unlike acylated myocardial CD59 (CD59H) neither CD59U nor PIPLC-treated CD59H became incorporated into poly-C9 complexes, suggesting that the interaction of CD59 with C9 requires the lipid anchor. Human C9 and heterologous C9 from guinea pig serum, as well as the YTH53.1 anti-CD59 mAb, inhibited the binding of CD59U to SC5b-8. On the other hand, soluble CD59U did not inhibit binding of C9 to SC5b-8, although CD59E has been shown to limit the number of C9 molecules entering into MAC. This suggests that two interaction sites between C5b-8 and C9 exist: one conferring the initial binding of C9 into the C5b-8 complex, and a second directing the insertion of C9 into the lipid bilayer. The latter interaction is the prerequisite for C9 polymerization and the target for interference by CD59.

Animals↗
PubMed1984

On the cause and nature of C9-related heterogeneity of terminal complement complexes generated on target erythrocytes through the action of whole serum.

The binding of C8 and C9 from human serum to target erythrocytes was quantified, and the molecular stoichiometries of C9:C8 within terminal C5b-9(m) complexes were determined. Low doses of serum generated terminal complexes with mean C9:C8 ratios of 2 to 3:1, whereas complexes generated by highest serum doses harbored an average of six to eight C9/C8 molecules. From the collective biochemical and ultrastructural data, we concluded that heterogeneous populations of terminal complexes regularly form on target membranes; those containing high numbers of C9 molecules (greater than or equal to six to eight) exhibit the structure of the classical "lesion", whereas those containing low numbers of C9 do not exhibit this typical structure, although they probably still function as small pores. A major cause for this heterogeneity of the lesions derives from shortage of C9, which is naturally present in a 2 to 1 molar ratio relative to C8 in serum. Generation of terminal complexes harboring high numbers of C9 on erythrocyte membranes is possible in spite of this natural shortage because SC5b-9 does not form in the fluid phase to compete for C9 binding. If interrupted, the process of C9-C9 oligomerization cannot be recontinued, and "incomplete" C5b-9 complexes are unable to bind additional C9 upon reincubation with this component. The demonstrated heterogeneity of terminal complexes with respect to their C9 content may explain the functional heterogeneity of complement lesions observed previously by other investigators.

Animals↗
PubMed1991

Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles.

During sublytic complement attack on human neutrophils, plasma-membrane vesicles are shed from the cell surface as a cell-protection mechanism. By using surface-iodinated neutrophils it was found that less than 2% of surface label was recovered in shed vesicles under conditions where 40% of complement component C9 was shed. SDS/PAGE of 125I-labelled shed vesicles and plasma membranes showed differences in iodination pattern, demonstrating the sorting of membrane proteins into the shed vesicles. Analysis of 32P-labelled phospholipids after labeling of neutrophils with [32P]Pi before sublytic complement attack showed the presence of phosphatidic acid, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and polyphosphoinositides in shed vesicles. Quantitative analysis using [3H]acetic anhydride-labelling method showed that the molar proportions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were the same in shed vesicles as in plasma membranes. In contrast, the molar proportions of cholesterol and diacylglycerol relative to sphingomyelin were almost twice those found in plasma membranes. The data demonstrate the existence of protein and lipid sorting mechanisms during the formation of shed vesicles when neutrophils are subject to sublytic complement attack. The term 'ectocytosis' is proposed to describe triggered shedding of right-side-out membrane vesicles from the surface of eukaryotic cells.

Autoradiography↗
PubMed1992

Regulation of complement functional efficiency by histidine-rich glycoprotein.

The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.

Animals↗
PubMed1987

Double immunofluorescence studies of IgA and poly C9 (MAC) in glomeruli from patients with IgA nephropathy.

Double immunofluorescent studies on IgA, poly (MAC) or C3 in glomeruli from patients with IgA nephropathy are described. Renal biopsy specimens were obtained from 12 patients with IgA nephropathy, four patients with proliferative glomerulonephritis (PGN) and two normal human kidney (NHK). These specimens were incubated with monoclonal anti-poly C9 (membrane attack complex; MAC) and then stained with FITC-labelled goat anti-mouse immunoglobulin (Ig) antiserum. After washing with phosphate buffered saline (PBS) (pH 7.4), the sections were stained with rhodamine-labelled rabbit anti-human IgA antiserum and examined by fluorescence microscopy. The sections were also stained with FITC-labelled goat anti-human C3 antiserum and then stained with rhodamine-labelled rabbit anti-human IgA antiserum. Markedly combined depositions of IgA and poly C9 or C3 in glomeruli were observed in patients with IgA nephropathy. There was a significant correlation between the deposition of poly C9 and the grading of histopathological injuries in such patients. There was also a significant correlation between the deposition of poly C9 in the extraglomerular vascular vessels and the ageing in patients with IgA nephropathy, PGN and NHK. It appears that the deposition of poly C9 might detect directly the activities of complement in glomeruli from patients with IgA nephropathy.

Age Factors↗
PubMed1990

The homologous restriction factor is immunologically related to complement components C8 and C9 and to lymphocyte pore-forming protein perforin through cysteine-rich domains.

The 65 kDa C8-binding protein or homologous restriction factor (C8bp/HRF) protects cells from complement (C)-mediated lysis by binding to C8 and abrogating lytic channel formation. Human C8bp/HRF is shown here to be immunologically related to human C8 and C9 and to murine lymphocyte poreforming protein (PFP, perforin). Polyclonal antibodies raised against purified C8, C9 and perforin react with C8bp/HRF. The antigenic epitopes shared by these four proteins are limited to cysteine-rich or disultide bridge-masked domains. Only complement proteins or perforin that have been disulfide-reduced elicit the production of cross-reactive antibodies when used as immunogens. Analogously, only C8bp/HRF that has been disulfide-reduced reacts with these antibodies. These results suggest that C8bp/HRF may belong to the complement/perforin supergene family. The function of homologous domains shared by these four proteins remains to be elucidated.

Animals↗
PubMed1985

Monoclonal antibodies recognizing a neoantigen of poly(C9) detect the human terminal complement complex in tissue and plasma.

The terminal complement complex (TCC), consisting of C5b, C6, C7, C8, and C9, contains neoantigens that are absent from the individual native components. Neoantigens are present both in the membrane-bound (MAC) and the fluid-phase (SC5b-9) complex. The present study describes production of monoclonal antibodies against neoantigens of both forms of the TCC. A convenient screening and detection system, based mainly on enzyme-linked immunosorbent assays, crossed immunoelectrophoresis with autoradiography, and affinity chromatography with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis including immunoblotting, is described in detail. Two monoclonal antibodies were specific for a neoantigen located in the poly(C9) moiety of the TCC. One of these antibodies, MCaE11, was used for immunohistochemical detection of MAC in tissue and for quantification of the fluid-phase TCC in ethylenediaminetetraacetic acid plasma.

Antibodies, Monoclonal↗
PubMed1991

DNA polymorphisms and linkage relationship of the human complement component C6, C7, and C9 genes.

In this report we describe the linkage between genes encoding human complement components C6, C7, and C9. Polymorphisms have been described at the DNA level for the C7 and C9 genes. We have studied 20 individuals by Southern blot analysis with four C6 cDNA subclones to detect restriction fragment length polymorphisms (RFLPs). We have found a Taq I polymorphism defined by two alleles of 8.0 (C6 H) and 6.0 (C6 L) kilobases (kb). RFLP segregation for the C6, C7, and C9 loci in informative families allowed us to estimate the maximum Lod scores at a recombination fraction of theta = 0.0 (C6-C7), theta = 0.0 (C7-C9), and theta = 0.0 (C6-C9). Significant linkage disequilibrium was found between C6 and C7 and between C7 and C9 loci in directly determined haplotypes of unrelated parents. Data from this study show that the genes encoding the human terminal complement components C6, C7, and C9 define a cluster in the short arm of chromosome 5. We propose that the clusters involving the C8A and C8B and the C6, C7, and C9 genes be referred to as MACI and MACII, respectively.

Chromosome Mapping↗
PubMed1993

Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.

In the activated complement system, vitronectin (complement S-protein) occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin-binding region of vitronectin and homologous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348-379 of the heparin-binding region, and a heparin-affinity polypeptide, protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b-7 membrane attachment, but was a potent inhibitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation takes place through the heparin-binding region.

Amino Acid Sequence↗
PubMed1985

Measurement of C9 concentrations using an immunochemiluminometric assay.

A 2-site immunochemiluminometric assay is described for the measurement of complement component C9 concentrations in biological fluids as an aid in the diagnosis and management of immune-based diseases. The assay utilises 2 monoclonal antibodies one of which is labelled with a chemiluminescent acridinium ester and one of which is covalently coupled to reprecipitated aminoaryl cellulose. The incubation time is 1 h with simultaneous reagent addition. The working range of the assay is 10-2500 micrograms/1 at CVs less than or equal to 10% and the results are in excellent agreement with those of an immunoradiometric assay. The assay exhibits superior performance to other immunochemical and immunoassay techniques and has the advantage of using stable, non-radioactive reagents.

Antibodies, Monoclonal↗
PubMed1977

Deviated lysis (d.l.): III. Kinetics of interaction of d.l. activity with chicken erythrocytes: evidence for E formation.

The interaction of d.l. activity with chicken red cells (CE) generates a cell intermediate with the properties of classical E*. Generation of CE* by d.l. activity at 37 degrees C is rapid, while there is a considerable lag in the conversion of CE* to ghost and hemoglobin. Conversion of CE* to ghosts can be blocked by high concentration of EDTA and/or 0 degrees C. CE* contain at least C6 and C9 on their surface.

Animals↗
PubMed1994

Formation of the terminal complement complex on agarose beads: further evidence that vitronectin (complement S-protein) inhibits C9 polymerization.

Vitronectin occupies the metastable binding site of C5b-7, which is unable to insert membranes as part of the complement lytic attack. Some evidence has been presented that vitronectin inhibits also membrane-associated pore formation by inhibiting C9 polymerization in the terminal complement complex (TCC). The authors wished to add to this background by studying the effect of vitronectin on formation of TCC on a carbohydrate surface like agarose beads, an alternative complement pathway activator. Bound TCC was detected by monoclonal and polyclonal antibodies to C9-neoepitopes. Soluble SC5b-7 and TCC (SC5b-9) did not bind to the agarose beads. Using serum or isolated complement factors for the alternative and terminal pathways, the authors found that vitronectin reduced the density of C9-neoepitopes on the beads. As there was no convincing evidence for association of vitronectin with the factors C5b-8 of the agarose-bound TCC, it was concluded that vitronectin bound directly to C9 in TCC and inhibited C9 polymerization within the complex. The authors have shown that TCC can bind to a carbohydrate surface like agarose (an alternating polymer of galactose moieties) in the absence of lipid. These results suggest that vitronectin can limit the lytic effect of membrane-bound TCC by inhibiting C9 polymerization.

Animals↗
PubMed1993

Antisense sequences of 20-kDa homologous restriction factor (HRF20) are found in C9 and the C8 beta chain of homologous complement.

We examined, on the basis of the fact that sense and antisense peptides have affinity for each other, whether any relationship exists between homologous restriction factor (HRF20), a membrane inhibitor of the terminal stage of homologous complement attack, and the antisense sequence of the terminal complement components C8 and C9. In this article, we demonstrate that there are two regions of C9 that contain antisense sequences to one continuous region of HRF20 and that this relationship exists between human HRF20 and human C9, but not mouse C9. We also found one region of the C8 beta chain that contains an antisense sequence to HRF20.

Amino Acid Sequence↗
PubMed1986

Human alveolar macrophages synthesize the functional alternative pathway of complement and active C5 and C9 in vitro.

Attachment of protein to agarose beads cultured with macrophages in protein-free medium containing 3H-leucine, shows that de novo synthesis of protein with affinity to the beads takes place. We also found that monoclonal antibodies against human C3c, C3g, and a C9-neoantigen as well as polyclonal antibodies against human C5 and C9, bound to agarose beads that had been kept with the macrophage cultures. Demonstration of C3 derivatives on the agarose beads shows that the essential complement factors of the alternative pathway are synthesized and have been activated by the beads. Deposition of C5 and the detection of a neoantigen of C9 on the beads, indicates that the whole terminal complement pathway has been formed and activated. We conclude that human alveolar macrophages form in vitro the functional alternative pathway of complement, C5 and C9, and we have indirect evidence for synthesis of C6, C7, and C8.

Antibodies, Monoclonal↗