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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↗
PubMed1981

Large scale isolation of functionally active components of the human complement system.

In the present work a scheme is presented for the isolation of multiple components of human complement in a functionally and biochemically pure state and with full hemolytic activity. These preparative procedures allow high recovery of milligram and gram quantities of particular complement components from a large pool (2-11 liters) of fresh EDTA plasma in no more than four chromatographic steps. Many components (C3bINA, C5, C3, C1EI, C4, and C9) are recovered functionally pure or highly purified following the first chromatographic step employing DEAE-Sephacel and may be utilized as reagents with no further purification. Prior to anion exchange, individual units of plasma are treated with inhibitors of complement activation and serum proteases, the pooled plasma is fractionated with polyethylene glycol, depleted of plasminogen on Sepharose-lysine, and rapidly ultrafiltered to low ionic strength and high protein concentration. The high degree of resolution of the components on DEAE-Sephacel subsequently obtained is demonstrated by the functional recovery and purification in a representative experiment as indicated (in their order of elution) for the following proteins: C3bINA (24%, 18-fold), C2 (74%, 12-fold), C7 (87%, 14-fold), factor B (55%, 8.7-fold),, C8 (50%, 16-fold), C6 (82%, 25-fold), beta 1H (39%, 12-fold), C5 (62%, 111-fold), C3 (99%, 64-fold), C1EI (42%, 135-fold), C9 (80%, 297-fold), and c4 (78%, 164-fold). Other components separated by these procedures include C1q and C4 binding protein. Additional steps described, which demonstrate the utility and effectiveness of this preparative scheme, have allowed isolation of C3, C5, and C7 as pure components with full hemolytic activity as judged by functional, immunochemical, and physicochemical criteria. C8, also isolated as a homogeneous protein, was recovered with partial hemolytic activity. All these components were recovered in high yield and in the purification as indicated: C3 (61%, 103-fold), C5 (24% 1350-fold), C7 (19%, 2260-fold), and C8 (32%, 547-fold). Complement components C6, beta 1H, factor B, and C2 in addition to C3bINA, C1EI, C4, and C9 are recovered partially purified with good activity and are amenable to further purification.

Complement C3↗
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↗
PubMed1995

Delineation of additional genetic bases for C8 beta deficiency. Prevalence of null alleles and predominance of C-->T transition in their genesis.

We studied the molecular bases for C8 beta deficiency in 34 unrelated families from the United States and the former Soviet Union. These families represented 69 unrelated null alleles of which 59 (86%) were found to be due to a previously described C-->T transition in exon 9. Six additional null alleles were also caused by C-->T transitions, of which four (6%) were located at base 388 in exon 3, one (2%) at base 298 in exon 3, and one (2%) involved cytosine 847 in exon 6. All of the null alleles affecting cytosine 388 were linked to the sequence polymorphism at base 376, which determines the uncommon C8 beta acidic allotype. Two null alleles were caused by single base pair deletions of cytosines at positions 430 and 632 in exons 3 and 5, respectively. Of the characterized null alleles, 97% were due to C-->T transitions in which an arginine (64 alleles) or a glutamine (one allele) was replaced by a stop codon. The basis for this apparent high frequency of C-->T transitions occurring in a relatively short stretch of DNA is uncertain.

Alleles↗
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↗
PubMed1994

[Clinical features and diagnosis of paroxysmal nocturnal hemoglobinuria: correlates with the deficiency of GPI-anchored membrane proteins].

Glycosyl phosphatidylinositol (GPI)-anchored membrane proteins are deficient in the blood cells affected by paroxysmal nocturnal hemoglobinuria (PNH). The relation of the deficiencies of CD59 and CD14 with the clinical features of PNH are reported. CD59 binds to complement components C8 and C9 derived from human sera and inhibits the C5b-9-mediated hemolysis in a species-selective manner. The CD59-binding sites were revealed to be localized in the alpha subunit of C8 and in the "b" domain of C9 (thrombin fragment). The deficiency of CD59 in PNH is causatively related to the hemolytic features in PNH. A monocyte differentiation antigen, CD14, is deficient in the affected PNH monocytes. CD14 is reported to be one of the receptors to lipopolysaccharide (LPS). LPS-binding to the monocytes were revealed to be mediated through CD14 on monocytes. Enhancement of LPS-binding to monocytes by the presence of serum was not seen to PNH-affected monocytes. PNH-affected monocytes showed impaired TNF-alpha production in response to LPS. The deficiency of CD14 indicates the abnormality in PNH-affected monocytes, however, its significance in the clinical features of PNH is to be clarified.

Antigens, CD↗
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↗
PubMed1991

Secondary structure-based profiles: use of structure-conserving scoring tables in searching protein sequence databases for structural similarities.

The profile method, for detecting distantly related proteins by sequence comparison, has been extended to incorporate secondary structure information from known X-ray structures. The sequence of a known structure is aligned to sequences of other members of a given folding class. From the known structure, the secondary structure (alpha-helix, beta-strand or "other") is assigned to each position of the aligned sequences. As in the standard profile method, a position-dependent scoring table, termed a profile, is calculated from the aligned sequences. However, rather than using the standard Dayhoff mutation table in calculating the profile, we use distinct amino acid mutation tables for residues in alpha-helices, beta-strands or other secondary structures to calculate the profile. In addition, we also distinguish between internal and external residues. With this new secondary structure-based profile method, we created a profile for eight-stranded, antiparallel beta barrels of the insecticyanin folding class. It is based on the sequences of retinol-binding protein, insecticyanin and beta-lactoglobulin. Scanning the sequence database with this profile, it was possible to detect the sequence of avidin. The structure of streptavidin is known, and it appears to be distantly related to the antiparallel beta barrels. Also detected is the sequence of complement component C8, which we therefore predict to be a member of this folding class.

Amino Acid Sequence↗
PubMed1995

Molecular mapping of SSRs for Pgm1 and C8b in the vicinity of the rat fatty locus.

Recessive mutations at the rat fatty locus (fa, facp), which produce obesity, insulin resistance, and diabetes, provide useful experimental models for similar phenotypes in humans. The molecular pathogenesis of the metabolic phenotype in animals segregating for fa is unknown and difficult to study once the confounding metabolic effects of obesity are present. Although various experimental methods distinguish preobese from lean rats (phenotypic markers and molecular markers genetically linked to fatty), technical difficulties limit their utility. We report the identification of two (GT)n simple sequence repeats (SSRs) near the rat phosphoglucomutase gene (Pgm1) gene and two SSRs, (GA)n and (GT)n, near the rat complement component 8 beta gene (C8b). These SSRs map to an approximately 4-cM interval flanking the fatty locus on rat chromosome 5. Use of these molecular markers in combination offers an improved method for early assessment of gene dosage for fa and hence for studying the fundamental molecular physiology underlying the derangements of metabolism and behavior resulting from mutations in this gene.

Alleles↗
PubMed1986

Genetic polymorphism of human complement component C81 in the Japanese population.

Genetic polymorphism of human C81 has been investigated using polyacrylamide gel isoelectric focusing (PAGIEF) in the presence of 3.1 M urea followed by electroblotting with enzyme immunoassay. In 448 individuals phenotypes of C81 were classified into three common and four rare patterns, and these were considered to be controlled by two common alleles, C81 A and C81 B, and three rare alleles which were tentatively designated C81 A1J and C81 A2J for acidic variants and C81 B1J for the basic variant. The alleles of C81 A2J and C81 B1J are new rare alleles, but C81 A1J might correspond to C81 A1 in the former studies. Family data were in accordance with the hereditary rules. The gene frequencies were estimated as C81 A is 0.6228, C81 B is 0.3672, C81 A1J is 0.0078, C81 A2J is 0.0011, and C81 B1J is 0.0011, respectively. The gene frequencies of the two common alleles agreed approximately with other ethnic groups. PAGIEF of neuraminidase-treated plasma samples followed by electroblotting with enzyme immunoassay is applicable to the study of heterogeneity of C81.

Alleles↗