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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↗
PubMed1985

Effect of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential.

The carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide has been used to investigate changes in membrane potential (Em) which occur upon binding of complement proteins C5b-9 to the plasma membrane of blood platelets. Gel-filtered platelets exposed to C5b6 and C7 in serum-free medium show no change in Em from that of controls, as indicated by either 3,3,'-dipropylthiodicarbocyanine iodide fluorescence or by the distribution of [14C]tetraphenylphosphonium bromide. Addition of complement proteins C8 and C9 to the C5b67 platelets results in partial depolarization of Em, which spontaneously repolarizes to basal levels within 15-20 min at 37 degrees C. Under these conditions, C5b-9-treated platelets show no increase in lysis over complement-free controls. Isotonic replacement of external sodium by either potassium or choline alters both the rate and extent of membrane depolarization and inhibits the platelets' capacity to repolarize after C5b-9 assembly. Repolarization of Em to basal levels is also completely blocked by addition of ouabain, confirming that this recovery is mediated by the plasma membrane Na+/K+ pump. These results demonstrate that membrane binding of the C5b-9 proteins can induce a transient change in Em when bound to the plasma membrane at a sublytic concentration, providing a mechanism for target cell activation by these potentially cytolytic proteins.

Adult↗
PubMed1991

Reduced C8 beta messenger RNA expression in families with hereditary C8 beta deficiency.

Individuals with functional C8 beta deficiency are at increased risk for systemic neisserial infections. Studies by others have shown that the structural gene for this protein appears intact in deficient individuals. We studied affected individuals from 10 unrelated families to determine the basis for their defect. Using chain-specific antisera, C8 beta was undetectable on immunoblots of their sera. The polymerase chain reaction was used to probe cDNA synthesized from RNA isolated from human liver cells, HepG2 cells, peripheral blood monocytes, and fibroblasts to identify a readily available cell source expressing C8 beta message. Cells from each of these sources expressed C8 beta message. The identity of the amplified product was confirmed and this approach was used to probe cDNA synthesized from RNA harvested from monocytes or fibroblasts obtained from two unrelated families with C8 beta deficiency. C8 beta mRNA was readily detectable in C8 beta sufficient and heterozygous family members but required Southern blotting and hybridization to the 32P-labeled C8 beta probe for detection in the homozygous deficient probands. These results suggest that C8 beta-deficient individuals produce less C8 beta-specific mRNA than do normals and that the underlying basis for this deficiency is an abnormality in intracellular events that precede secretion.

Complement C8↗
PubMed1976

The membrane attack mechanism of complement: the three polypeptide chain structure of the eigth component (C8).

The purification of human C8 in milligram quantities from outdated human serum was achieved by ammonium sulfate precipitation (37.5-50% saturation) and ion exchange column chromatography employing CM-32 cellulose and QAE-Sephadex. The yield of C8 activity ranged from 2-9%, and the average purification was 1,700-fold. Fully reduced C8 was shown by SDS polyacrylamide gel electrophoresis to have three polypeptide chains which were present in equimolor ratios: alpha, 77,000 daltons; beta, 63,000 daltons; and gamma, 13,700 daltons. C8 denaturation by SDS and urea in the absence of reducing agents revealed two noncovalently linked subunits: alpha-gamma, 99,000 daltons, and beta, 75,000 daltons.

Complement C8↗
PubMed1986

Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes.

Complement effects on human polymorphonuclear leukocytes (PMN) have generally been ascribed to the anaphylatoxin C5a, which induces degranulation, superoxide anion generation, migration, and cell aggregation via interaction with membrane receptors. We here report that complement activation on the surface of antibody-sensitized human PMN provokes generation of the potent lipid mediator leukotriene B4 (LTB4) in strict dependence on complement component C8, but in the absence of detectable C9. The kinetics of LT generation are rapid, comparable with those observed after challenge with the calcium-ionophore A23187. LTB4 release is a distinct event that is dissociable from cytotoxicity as assessed by lactate dehydrogenase (LDH) release (dependent on C9) and from superoxide generation (independent of C8 and C9). It is dose dependent on extracellular calcium and is not observed in the absence of calcium. It is inhibited by substances interfering with calcium-calmodulin function (trifluoperazine and W7), but not by blockers of physiologic calcium channels (nimodipine, verapamil, and D 888). Addition of purified C8 to cells bearing C5b-7 induces a severalfold increase in their passive permeability to 45calcium. Sieving experiments with the use of marker molecules of different sizes collectively indicate the existence of small hydrophilic channels consisting exclusively or predominantly of C5b-8 complexes, which allow passive transmembrane flux of small molecules with Mr less than 200. Thus, noncytolytic terminal complement complexes may serve as a biological bypass gate for calcium in PMN membranes, triggering the arachidonic acid cascade with generation of LTB4 at doses well below the threshold required to invoke overt cell damage.

Calcium↗
PubMed1989

Local complement activation, thromboxane-mediated vasoconstriction, and vascular leakage in isolated lungs. Role of the terminal complement sequence.

In situ complement activation was induced in isolated, ventilated, and blood-free perfused rabbit lungs. Six to 11% human serum (vol/vol) was repeatedly admixed to the recirculating Krebs Henseleit buffer during 10-min periods, interrupted by rinsing phases with serum-free medium. This caused dose-dependent, reversible, and reproducible pulmonary artery pressor responses with pressure peaks or plateaus of 8 to 22 mm Hg. Pressor responses were paralleled by a marked release of thromboxane (TxA2) and prostaglandin I2 into the medium. Heat-inactivated serum, purified fluid-phase SC5b-9, and serum that had been activated with inulin in the absence or presence of a carboxypeptidase inhibitor all failed to elicit pressor responses. Both pressor response and prostanoid release were strictly dependent on complement factor C8, and evidence for C8 binding was obtained with the use of 125I-C8. Inhibitor studies collectively indicated that TxA2 was the predominant vasoconstrictive agent. Although repetitive short-term application of human serum induced no major lung weight gain, prolonged exposure to serum (40 to 80 min) without intermittent rinsing phases caused a delayed increase in the capillary filtration coefficient to maximally 3- to 4-fold values, with marked lung edema formation. We conclude that in situ activation of complement in the rabbit lung vasculature causes immediate intrapulmonary prostanoid generation and thromboxane-mediated vasoconstriction, independent of circulating leukocytes, but dependent on the formation of terminal membrane-complement complexes. Continuous complement activation results in a delayed increase in lung vascular permeability.

Animals↗
PubMed1990

Chromosomal assignment and physical linkage of the human C8 loci: implications regarding C8 polymorphisms and deficiencies.

cDNA probes specific for the alpha, beta and gamma subunits of human C8 were used to establish that genes encoding alpha and beta are physically linked and in a 5' alpha-beta 3' orientation on chromosome 1. The gene encoding the gamma subunit was mapped to chromosome 9q. These results explain the close genetic linkage between alpha-gamma and beta on chromosome 1 and indicate that alpha-gamma polymorphisms detected in earlier genetic studies are determined solely by alpha. Results summarized in this report were presented at the VIth Complement Genetics Workshop and Conference (Mainz, FRG, 1989).

Chromosome Mapping↗
PubMed1992

Linkage and association studies with C8A and C8B RFLPs on chromosome 1.

Linkage relations for the C8A and C8B BamHI RFLPs have been investigated. A peak lod score of 4.52 at recombination fraction zero was obtained between the two C8 genes. Combined with our previously obtained linkage data (Rogde et al. 1986) the maximum lod score is 7.53 at recombination fraction zero. The compiled C8-PGM1 linkage data from this and the previous study gave a maximum lod score of 22.02 at recombination fraction 0.11 (0.07-0.16) with no sex difference. A chromosome 1p reference marker, D1S57, has been applied in this linkage study. A maximum lod score of 5.06 between the C8 cluster and D1S57 at theta = 0.18 (0.11-0.28) was recorded. The linkage analyses and triply informative families gave evidence that the C8 loci are situated about halfway between PGM1 and D1S57 on the short arm of chromosome 1. There was no evidence of allelic association between the C8A and C8B BamHI RFLPs in 62 unrelated haplotypes.

Chromosome Mapping↗
PubMed1986

Human C81 (alpha-gamma) polymorphism: detection in the alpha-gamma subunit on SDS-PAGE, formal genetics and linkage relationship.

The molecular basis of human C81 (alpha-gamma) polymorphism could be elucidated by immunoprecipitation of human C81 allotypes and separation of the alpha-gamma and beta subunits on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. If the C8 molecules were completely reduced, C81 polymorphism was no longer detectable on SDS-PAGE. It is concluded that C81 variation depends on charge rather than molecular weight differences. Four C81 allotypes, the common A and B and two rare allotypes provisionally named A2 and B1, could be distinguished. The rare allotype A1 as detected by isoelectric focusing with subsequent C8 (alpha-gamma)-dependent functional overlay could no longer be visualized on SDS-PAGE. This allotype may therefore be elicited only in the intact C8 molecule. The beta-chain polymorphism named C82, probably also reflecting charge variation of the C8 molecule, could not be detected yet on SDS-PAGE. The distributions of C81 phenotypes and their respective allele frequencies were in good agreement with previously reported data. In the study of 30 families with 100 offspring, no deviation from the rule of at least four codominant alleles at one genetic locus was found. Linkage between C81 gene(s) and PGM1a encoded on chromosome 1 could be confirmed. The following estimates were obtained: (formula; see text) with S theta being the standard error of the maximum likelihood estimate theta. The new technique for allotyping human C81 at the subunit may provide a new tool for the differentiation of qualitative and quantitative variation of the eighth component of human complement.

Alleles↗
PubMed1977

On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes.

The preceding paper (Hammer, C.H., A. Nicholson, and M. M. Mayer, 1975, Proc. Natl. Acad. Sci., 72:5076) presented evidence on insertion of polypeptide chains from the C5b and C7 subunits of C5b, 6, 7 complex into the phospholipid bilayer of erythrocyte membranes. In the present study, EAC1-8 and EAC1-9 (sheep erythrocytes carrying rabbit antibody and complement proteins C1 through C8 or C9, respectively), prepared with either 125I-C8 or 125I-C9, were incubated with trypsin or chymotrypsin and the release of 125I was measured. Only 9 to 19% of the specifically bound radioactivity was released. In addition, elution experiments were performed with 0.02 M EDTA-1.0 M NaCl. This solution did not elute C9 from EAC1-9. By contrast cellbound C9 was recovered from erythrocyte membranes with sodium dodecyl sulfate (SDS). Thus, enzymatic stripping and elution experiments indicate that cellbound C9 behaves like an integral membrane protein, presumably due to insertion into the lipid bilayer. EAC1-9 membranes that had been subjected to extended digestion with trypsin or chymotrypsin were extracted with SDS to recover the enzyme-resistant part of the C9 molecule from the membrane. Even though this domain of C9 carried 90% of the radioiodine associated with native C9, its m.w. was found to be only 18,000 daltons by analysis on SDS-PAGE. This represents one-quarter of the native C9 molecule.

Animals↗
PubMed1990

C8 reference typing report and nomenclature recommendation.

Using two different typing techniques (i.e. polyacrylamide gel isoelectric focusing (PAGIF) with Western blot and SDS-polyacrylamide gel electrophoresis of precipitated C8 under nonreducing conditions with Western blot), the following observations were made during the reference typing for C81 (C8A). The Japanese variant A1J is probably identical with A1Cauc, whereas B1J is definitely different from B1Cauc and could therefore provisionally be named HB3'. Variant 'A2' from Japan is focused in an intermediate position, but different from M1 and could be named 'M2'. Both variants possess normal A subunits. B2 from Japan is clearly different from B1Cauc and should retain its designation. In PAGIF, its subunit has a position more anodal than A. Summarizing these results, more information is needed before a final nomenclature can be proposed.

Blotting, Western↗
PubMed1989

C8A and C8B polymorphisms in Norwegians and Norwegian lapps.

C8 inheritance patterns in 364 mother-child pairs formed the basis for evaluation of the existence of silent alleles (null alleles) in the genes determining the two known polymorphic C8 systems. While evidence for such alleles was not found in C8A (alpha-gamma complex), two observations of null allele segregation in C8B (beta chain) indicate a C8BQ*0 allele frequency of about 0.07. Two population samples comprising 150 Lappish and 1,264 non-Lappish Norwegians were examined for phenotype distributions in C8A and C8B. The phenotype distributions were mainly in accordance with the expected Hardy-Weinberg distribution. The results for C8A indicated simple, codominant inheritance of two frequent and several rare alleles. Allele frequencies were similar in the two populations. The C8A B gene frequency in Norwegians was significantly lower than that in FRG and higher than that in Negroes. C8B allele frequencies were also calculated from gene counts in the population material, but with due corrections for the C8BQ*0 frequency observed in the mother-child material.

Alleles↗
PubMed1986

Number of hits necessary for complement-mediated hemolysis.

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1-7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, the human C8 each followed a one-hit mechanism, while that of human C9 showed ka multi-hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one-hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9-deficient human serum was used for lysis of EA, multiple-hit curves were obtained. The mechanism of lysis by C5b-8 may differ from that by C5b-9.

Animals↗
PubMed1986

Bactericidal-activities of human polymorphonuclear leukocyte proteins against Escherichia coli O111:B4 coated with C5 or C8.

The postnuclear supernatant of disrupted polymorphonuclear leukocytes exhibited bactericidal activity on Escherichia coli O111:B4 coated with immunoglobulin M antibodies and C5 or C8 but not on C3- or C7-coated bacteria. To characterize this antimicrobial activity further, granules obtained from the postnuclear supernatant were extracted with sodium acetate (pH 4) and the soluble extract was subsequently fractionated through carboxymethyl cellulose and Sephacryl S-200. Over 90% of the activity present in the starting material was recovered in the soluble granule extract. Kinetic and dose-response analyses of the bacterial activity of the polymorphonuclear leukocyte extract on BAC1-5 and BAC1-8 revealed different susceptibilities to killing of these two bacterial intermediates; they also differed for their susceptibilities to killing at 37 degrees C and at room temperature. The suggestion raised by these data, that BAC1-5 and BAC1-8 could be killed by different bactericidal factors, was confirmed by the findings that separate fractions of the soluble granule extract obtained by carboxymethyl cellulose and Sephacryl S-200 chromatography exhibited specific activity on either BAC1-5 or BAC1-8, whereas other fractions were active on both intermediates.

Blood Bactericidal Activity↗
PubMed1988

The structure of human complement component C7 and the C5b-7 complex.

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.

Amino Acid Sequence↗