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PubMed1978

Differences in the terminal steps of complement lysis of normal and paroxysmal nocturnal hemoglobinuria red cells.

The number of microscopically visible lesions produced on the membrane for a given degree of lysis on normal cells as on PNH cells. Since complement lesions were not formed until C8 or C9 was incorporated into the complement sequence, the results suggest that increased lysis of red cells in PNH is due at least in part to more efficient penetration of the PNH membrane by the terminal lytic sequence of complement. Furthermore, the efficiency of the terminal lytic sequence in the lysis of PNH cells when complement was activated by the alternative pathway and the classical pathway was analyzed. There was no significant difference (p less than 0.01) in the number of lesion present at an equivalent degree of lysis when initiated by antibody, cobra venom factor, or acidification. Thus, the efficiency of the terminal lytic sequence does not vary with different modes of activation.

Complement C8↗
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↗
PubMed1988

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit.

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.

Antibodies, Monoclonal↗
PubMed1994

Structure of a soluble, glycosylated form of the human complement regulatory protein CD59.

BACKGROUND: CD59 is a cell-surface glycoprotein that protects host cells from complement-mediated lysis by binding to and preventing the normal functioning of the complement proteins C8 and/or C9 which form part of a membrane penetrating assembly called the membrane attack complex. CD59 has no structural similarity to other complement proteins, but is an example of a plasma protein domain type found also in murine Ly-6 proteins and the urokinase-type plasminogen activator receptor. RESULTS: CD59 was purified from human urine, retaining the N-glycan and at least some of the non-lipid component of the glycosylphosphatidylinositol membrane anchor. The three-dimensional structure of the protein component has been determined in the presence of the carbohydrate groups using two-dimensional NMR spectroscopy. The protein structure is well defined by the NMR data (root mean square deviation from the mean structure of 0.65 A for backbone atoms and no distance constraint violations greater than 0.4 A). Structure calculations were also carried out to model the orientation of the N-acetylglucosamine residue that is directly linked to Asn18. CONCLUSIONS: The main features of the protein structure are two antiparallel beta-sheets (a central one with three strands and another with two), a short helix that packs against the three-stranded beta-sheet, and a carboxy-terminal region that, although lacking regular secondary structure, is well defined and packs against the three-stranded beta-sheet, on the opposite face to the helix. We have used the structure, in combination with existing biochemical data, to identify residues that may be involved in C8 binding.

Amino Acid Sequence↗
PubMed1987

Complement complex C5b-8 induces PGI2 formation in cultured endothelial cells.

The effects of the terminal complement sequence on prostacyclin (PGI2) generation in antibody-sensitized pulmonary arterial endothelial cells were examined. Whereas C5b-7 complement complexes induced no PGI2 formation, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8-dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. In contrast, W7 [N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide] and trifluoperazine, inhibitors of calmodulin activity, all reduced the C5b-8-dependent PGI2 generation. None of the inhibitors used impaired Ca2+ flux into the cells. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six- to seven-fold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51Cr O4(2-), [3H] aminobutyric acid, and [3H]sucrose, but not for [3H]inulin and [3H]dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+ bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway.

Animals↗
PubMed1981

Recurrent bacterial meningitis associated with C8 and IgA deficiency.

A patient with recurrent bacterial meningitis who lacked circulating C8, serum IgA, and secretory IgA is reported. The combined absence of C8 and IgA deficiency has not been previously noted. Although the association of disseminated neisserial infection and absence of the terminal complement components is now well established, the deficiency of bactericidal capability as well as mucosal humoral defense may have been additive in predisposing the patient to recurrent meningitis. The pedigree was uninformative regarding possible linkage of these two uncommon genetic errors.

Adolescent↗
PubMed1986

Dysfunctional C8 beta chain in patients with C8 deficiency.

Two sera from unrelated individuals, each lacking C8 activity, were examined by Western blot analysis. Using antisera raised against whole C8, the two sera are shown to lack the C8 beta chain, indicating a C8 beta deficiency, which is frequently observed in cases of dysfunctional C8. In contrast, by means of a specific anti-C8-beta antiserum, a C8 beta-like polypeptide chain of apparently identical molecular weight compared to normal C8 beta was detected. Digestion of normal and dysfunctional C8 beta with Staphylococcus aureus V8 protease revealed distinct differences in the enzymatic digestion pattern. We conclude that the dysfunction in the C8 protein in these two patients resides in the dysfunctional C8 beta chain, and that this form of C8 deficiency is distinct from C8 deficiencies previously reported, in which one or both C8 subunits are lacking.

Adult↗
PubMed1984

Population and formal genetics of the human C81(alpha-gamma) polymorphism.

One hundred and ninety-six unrelated healthy individuals and 30 families with 75 offspring have been studied for the C81(alpha-gamma) polymorphism. The following allele frequencies were calculated: C81*A = 0.5536; C81*B = 0.4286; C81*A1 = 0.0178. Observed and expected phenotype frequencies were in a good agreement according to the Hardy Weinberg law. No exceptions from the mode of inheritance were found. In family W the segregation of the rare allele C81*A1 could be followed. Comparing the results of this study with previous data from Boston and Oslo, a combined technology including C8-dependent lysis and C8 structural variation is suggested for future investigations.

Alleles↗
PubMed1994

Effects of complement activation products on the synthesis of decay accelerating factor and membrane cofactor protein by human mesangial cells.

We previously demonstrated that activation of terminal complement components (C8 and/or C9) increases the synthesis and expression of decay accelerating factor (DAF) on human glomerular cells. DAF is a cell membrane-associated complement regulatory protein that inhibits complement activation on cell surfaces. In the present studies we evaluated, first, the mechanisms by which complement activation stimulates DAF synthesis, and second the effect of complement activation on the synthesis. and expression of membrane cofactor protein (MCP), another complement regulatory protein, by human mesangial cells (HMC) in culture. Complement activation by immune complexes resulted in increased DAF mRNA levels by at least two mechanisms: deposition of activated C3 on HMC and generation of soluble complement activation products, specifically C5a. The increase in DAF mRNA levels induced by activated C3 or C5a was short lived (less than 4 hr). In contrast, the up-regulation of DAF mRNA levels induced by activation of the complete complement cascade persisted for at least eight hours. The effect of complement activation on DAF mRNA levels was not affected by cycloheximide, a protein synthesis inhibitor. However, cycloheximide alone resulted in a significant up-regulation of DAF mRNA levels on HMC. In contrast to those findings complement activation did not cause an up-regulation of MCP mRNA, nor an increase in the synthesis of this protein. However, by FACS, complement produced a small but significant increase of MCP protein levels on HMC. In conclusion, both MCP and DAF are present on HMC. Several activated complement components are capable of increasing DAF mRNA levels, but DAF protein levels increase only after activation of the whole complement cascade.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1989

Terminal complement complex and endothelial cells.

The effects of the terminal complement sequence on two endothelial cell functions (prostacyclin (PGI2) generation and permeability of an endothelial cell monolayer) were examined in antibody-sensitized pulmonary artery endothelial cells. Whereas C5b-7 complement complexes induced on PGI2 formation and no enhanced endothelial permeability, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release and in a substantially increased permeability of an endothelial cell monolayer in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8 dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six to sevenfold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51CrO4(2-), 3H-aminobutyric acid, and 3H-sucrose, but not for 3H-inulin and 3H-dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+-bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway and the actin-myosin system.

Arachidonic Acid↗
PubMed1990

Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed.

Blotting, Western↗
PubMed1995

Complement component C7 is a plasminogen-binding protein.

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.

Autoradiography↗
PubMed1985

Inherited C8 beta subunit deficiency in a patient with recurrent meningococcal infections: in vivo functional kinetic analysis of C8.

A 16 year old with recurrent meningococcal infections is reported. Absence of haemolytic activity in both the classical and alternative pathways resulted from an absence of functional C8. Addition of functional C8 restored hemolytic activity. Antigenically deficient C8 was present in the serum and isoelectric focusing of serum confirmed the absence of the C8 beta chain. Following the infusion of fresh frozen plasma, we followed the decay in C8 functional activity as well as total haemolytic activity. C8 activity peaked at about 3 h with a half-life survival estimated to be 28 h. The kinetics of total haemolytic activity showed a slower decay with an exponential decline over 72 h and a half-life of 55 h. Fresh frozen plasma may be of value in the treatment of patients with C8 deficiency and acute Neisserial infections.

Adolescent↗
PubMed1976

Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.

Cell Membrane Permeability↗
PubMed1988

Differential functional expression of the C8 subunits. Primary role of C8 beta in assembly of intact C8.

The eighth component of C (C8) is composed of two subunits C8 beta and C8 alpha-gamma, which are non-covalently bound in a 1/1 ratio in the intact molecule. The genes encoding the polypeptide chains composing the subunits demonstrate close genetic linkage. To assess the functional expression of these genes at the protein level, normal human serum and C8-deficient sera were electrophoresed in native polyacrylamide gels following which C8, C8 beta, and C8 alpha-gamma were detected using hemolytic overlays. These experiments demonstrated that normal sera contained free C8 alpha-gamma in addition to intact C8. Free C8 alpha-gamma was not observed when C8 was reconstituted by mixing C8 beta-deficient serum with C8 alpha-gamma-deficient serum in a ratio optimized for C8 activity, suggesting that the free C8 alpha-gamma observed in normal serum was not due to dissociation of intact C8. Inasmuch as this technique did not adequately separate C8 and C8 beta, sera were also examined by anion exchange chromatography. C8 alpha-gamma-deficient serum contained C8 beta in a single peak in the 1.4 ms/cm fall through. C8 beta-deficient serum contained a major peak of C8 alpha-gamma at 7.1 ms/cm and a lesser peak coeluting with C9 at 9.5 ms/cm. Normal serum contained both intact C8 eluting between 2.4 to 5.5 ms/cm and C8 alpha-gamma eluting at 7.1 ms/cm. Free C8 beta was not detectable in normal serum indicating that free C8 alpha-gamma was not due to C8 dissociation. Mixing aliquots from the chromatographic peak of C8 beta activity with the peaks of C8 alpha-gamma activity in C8 beta-deficient serum or in normal serum generated intact C8 hemolytic activity. Non-reducing SDS-PAGE and Western blotting with anti-C8 confirmed the presence of antigenic material of appropriate m.w. in each peak. These findings demonstrate that serum contains excess C8 alpha-gamma relative to C8 beta, despite the equimolar presence of the subunits in intact C8. Thus the availability of C8 beta determines the quantity of C8 produced. Further, these data suggest the possibility that the C8 structural genes may be differentially expressed despite their close genetic linkage.

Blotting, Western↗