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PubMed1988

Molecular analysis of human complement component C5: localization of the structural gene to chromosome 9.

A human C5 clone (pC5HG2) was isolated from a cDNA library constructed from Hep G2 mRNA. The DNA sequence showed that the pC5HG2 insert was comprised of 3309 base pairs of pro-C5 coding sequence and 404 base pairs of 3'-untranslated sequence. The derived amino acid sequence contained the entire coding sequence of the C5 alpha-chain, the beta-alpha-chain junction region, and 100 amino acids (approximately 50%) of the beta-chain. Protein sequences of four C5 tryptic peptides were aligned exactly to this sequence and demonstrated that C5 synthesized and secreted by Hep G2 cells is probably identical with plasma-derived C5. Coding sequence alignment of the human C5 sequences with those of murine C5 indicated that 80% of the nucleotides and 79% of the amino acids were placed identically in the two species. Amino acid sequence alignment of the homologous family members C3, C4, and alpha 2-macroglobulin with that of C5 demonstrated 27%, 25%, and 19% identity, respectively. As was found in murine C5, the corresponding thiol ester region of human C5 contained several conserved amino acids, but the critical cysteine and glutamine residues which give rise to the intramolecular thiol ester bond in C3, C4, and alpha 2-macroglobulin were absent in C5, having been replaced by serine and alanine, respectively. With the use of a panel of hamster-human somatic cell hybrids, the C5 gene was mapped to human chromosome 9. In situ chromosomal hybridization studies employing metaphase cells further localized the gene to bands 9q32-34, with the largest cluster of grains at 9q34.1.

Amino Acid Sequence↗
PubMed1988

Localization of a hydrophobic domain in human C5.

The fifth component of human complement (C5), under physiological salt conditions, bound firmly to phenyl-Sepharose. This indicated the presence of hydrophobic sites on C5. These sites may play an important role in C5 haemolytic activity since this activity was inhibited by chaotropic ions (Mg2+, SCN-, I-) at relatively low concns (0.6-1 M). Charge shift experiments performed on purified C5 provided further evidence for the presence of hydrophobic sites within C5. Bidirectional charge shifts of C5 mobility were observed with the two detergent systems TX-100 + DOC and TX-100 + CTAB. In order to localize these sites, C5 was subjected to trypsin digestion. Three major fragments were evidenced by two-dimensional electrophoresis (agarose/SDS-PAGE and SDS-PAGE/SDS-PAGE), and were isolated by hydrophobic affinity chromatography. The first fragment, C5FI, was composed probably of a mixture of six alpha chain fragments (alpha 1-alpha 6) linked to the beta chain by disulphide bridges. The second fragment, C5FII, was composed of the beta chain linked to two alpha chain fragments: alpha 2 (Mr = 58 K) and alpha 4 (Mr = 32 K). The third fragment, C5FIII, contained two covalently linked chains (beta chain and alpha 4). The rank order for mol. wt, agarose gel electrophoretic mobility and hydrophobicity was respectively: C5 greater than or equal to C5FI greater than C5FII greater than C5FIII; C5 less than or equal to C5FI less than or equal to C5FII much less than C5FIII; and C5FII greater than or equal to C5 greater than C5FI much greater than C5FIII. From the relative hydrophobicity, the hydrophobic sites of C5 can be localized to the alpha 2 fragment (Mr = 58 K) since C5FIII lacked the alpha 2 fragment and was not hydrophobic. Cleavage and the liberation of the N-terminal part of C5 induced an increase in hydrophobicity of the remaining part of C5 (C5b and C5FII), suggesting a possible role of the hydrophobic sites in association of C5b with membranes.

Complement C5↗
PubMed1990

Combined complete C5 and partial C4 deficiency in humans: clinical consequences and complement-mediated functions in vitro.

A family is described with two siblings who suffered at different times from a single episode of meningococcal meningitis by Neisseria meningitidis groups B and C, respectively. In the two subjects, hemolytically active fifth component of complement (C5) was not detectable and antigenic C5 was less than 0.05% and less than 0.7% of normal, respectively. Repletion of sera by purified human C5 (70 micrograms/ml) restored total complement hemolytic activities. The asymptomatic first degree family members had C5 levels compatible with a heterozygous state of C5 deficiency. C4 allotyping revealed an inherited partial deficiency (Q0) of C4A and C4B in the family with a combined C4AQ0 and C4BQ0 heterozygous condition in one and C4BQ0 heterozygosity in the other C5 deficient (C5D) subject. To our knowledge, this is the first human kindred with recognized combined C5 and C4 deficiency. No other defect of the humoral and cellular immune system was found in this family, including specific immune response to tetravalent meningococcal vaccine. The effect of partial C4 deficiency on classical pathway function was assessed by inhibition of immune precipitation (IIP) of forming bovine serum albumin (BSA)/anti-BSA immune complexes. Sera from all family members showed normal IIP values, with exception of the subject with combined partial deficiency in C4A, C4B, and complete deficiency in C5. Despite undetectable functional C5 in the C5D sera, the titration of the alternative pathway indicated intact but deficient hemolytic activities when rabbit erythrocytes (EC) were used as indicator cells in the presence of Mg2+ and EGTA in an end-point or kinetic assay. Preincubation of the two sera at 0 degrees C for 60 min with rabbit ECs reduced alternative pathway hemolytic activity by 24 and 100%, respectively. When rabbit ECs were replaced by guinea pig ECs no alternative pathway function could be measured. The results indicate that the apparent functional activity of the alternative pathway in C5D sera strongly depends on a factor(s) present in such serum and/or on the detection system used. We conclude that the two C5D individuals of the family reported here may not have sufficient C5 activity to provide efficient protection against Neisserial infections in conditions where complement functions beyond C3 opsonic activity are required in vivo.

Adolescent↗
PubMed1986

Isolation, characterization and utilization of the fifth component (C5) of porcine complement.

The fifth component (C5) of porcine complement was isolated in immunochemically pure, homogeneous and native form. The isolated component was characterized, it has a molecular weight of 187,000 (under non-reducing conditions) and two non-identical chains of 112,000 and 76,000 under reducing conditions, respectively. The isolated C5 can be used for the detection of the C5b receptors on murine macrophages. Our findings are in good agreement with the results obtained using a rabbit and a human model, which suggests a high conservativeness of C5 during evolution. The described method of isolation of C5 from easily available porcine serum seems to be convenient for C5 receptors studies.

Animals↗
PubMed1992

[The association between deficiency of terminal complement components and the occurrence of meningococcal meningitis].

Seventeen patients with sporadic meningococcal meningitis, registered in Fukuoka city, were studied for complement deficiency. Four of the seventeen had a selective deficiency of the ninth component of the complement (C9), four had a deficiency of the seventh component of the complement (C7), and one had a deficiency of the fifth component of the complement (C5) as determined by both hemolytic and antigenic assay. Family studies suggested that these complement deficiencies in the present study were hereditary. Based on the incidence (0.036% of homozygous C9 deficiency in Fukuoka (171 C9 deficient individuals of 475, 886 blood donors), we have concluded that patients with C9 deficiency are more susceptible to meningococcal meningitis than normal individuals (p less than 0.001). In addition, the present study further supports that complement deficiency is common in patients with sporadic meningococcal meningitis.

Adolescent↗
PubMed1992

Activation of the fifth component of human complement by oxygen-derived free radicals, and by methionine oxidizing agents: a comparison.

The fifth component of human complement, C5, was activated by non-enzymical, chemical treatment in either of two ways: 1) by oxidation with a hydroxyl radical (OH.) generating system consisting of H2O2, FeEDTA, and ascorbate, activation product called C5(H2O2); 2) by oxidation with chloramine T, activation product called C5(Cl-T). Evaluating earlier findings, completed by new results, both products were compared. Both products are C5-like in that they are capable of binding C6 and form the nucleus for the cytotoxic complex C5-9. Both differ from C5b, the natural activation product of C5, as they comprise the whole, uncleaved C5 protein, and do not immediately decay when not bound to C6. In both cases the treatment involves oxidation of methionine residues in the C5 protein. However, while chloramine T specifically attacks only methionine, oxidation by the OH. generating system involves other amino acid residues, in addition. This probably explains the lower yield of C5b-like activity after treatment with H2O2, and other quantitative differences between C5(H2O2) and C5(Cl-T). Whereas the generation of C5(H2O2) may be physiologically relevant, C5(Cl-T) may prove to be a suitable object for the study of changes in the C5 molecule essential for its activation.

Chloramines↗
PubMed1979

C5 chemotactic fragment induces leukocyte production of tissue factor activity: a link between complement and coagulation.

Complement-activated human plasma causes generation of tissue factor in human leukocytes. This phenomenon appears to be related to the fifth component of complement (C5) as demonstrated by the use of C5 deficient-plasma and suppression of activity with antibody to C5. Isolation of the chemotactic factor from activated serum or trypsinization of purified C5 reproduces the phenomenon. These data provide evidence for a direct link between complement products and activation of the coagulation system. Because chemotactic peptides from C5 can be generated by a variety of enzymes, our findings suggest a relationship between complement, coagulation, and inflammation.

Blood Coagulation↗
PubMed1989

Measurement of complement activation in rabbit plasma or serum using monoclonal antibodies against C5a.

Eight murine monoclonal antibodies (MoAb) raised against the major zymosan-induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen-antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan-induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat-inactivated plasma. A cross-reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme-linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan-induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan-activated serum. MoAb 2A5E3 was able to bind 125I-labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen-antibody complexes, or cobra venom factor.

Animals↗
PubMed1988

Induction of an immune response to a self antigen.

The question has been addressed whether the endogenous B cell population of a mouse can be induced to secrete antibodies specific for a self antigen present in serum. The antigen studied was the fifth component of mouse complement (C5). Nude BALB/c mice which are C5 sufficient were used as a source of potentially C5-reactive B cells and endogenous serum C5 provided the antigenic stimulus. We purposely avoided immunization with C5 in adjuvant. T cells from C5-deficient mice which lack this component in serum and are therefore not tolerant of C5 were injected into nude mice as a source of T cell help for anti-C5 reactive B cells. Control groups received T cells from C5-sufficient euthymic donors, which are tolerant of C5. Initiation of a response to C5 was monitored by testing the hemolytic function of serum. Reduction of C5-dependent hemolysis was observed in sera of mice which had received T cells from C5-deficient donors. Recipients of T cells from C5-sufficient donors maintained normal hemolytic complement levels throughout the test period of 45 days. Reduction of functional complement levels correlated with the presence of immune complexes of anti-C5/C5. C5-specific antibodies were mainly IgG1 and carried the IgG1 allotype of BALB/c providing unequivocal evidence that they were derived from the endogenous B cell population of the C5-sufficient host.

Animals↗
PubMed1989

Mediator-induced activation of xanthine oxidase in endothelial cells.

Rat pulmonary artery endothelial cells incubated with human serum that has been complement-activated by addition of cobra venom factor reveal a pronounced conversion of xanthine dehydrogenase to xanthine oxidase. This process requires the availability of the fifth component of complement (C5) but not the presence of other components (C2 and C6-C9). The phenomenon can be reproduced by addition to endothelial cells of purified human recombinant C5a but not C5a desArg or C3a. The enzyme conversion process is relatively rapid (occurring within 5-10 min), requires the presence of intact endothelial cells, and does not require protein synthesis. Similar effects on endothelial cells have been obtained with human recombinant tumor necrosis factor alpha and the chemotactic peptide N-formyl-Met-Leu-Phe. In contrast, bradykinin, recombinant human interleukin 1 beta, and phorbol ester lack this biological activity. These findings suggest novel effects of inflammatory mediators on endothelial cells.

Animals↗
PubMed1985

Studies of human C5a as a mediator of inflammation in normal human skin.

C5a is an 11,000-D fragment of the fifth component of complement (C5) with potent anaphylatoxic and leukocyte chemotactic activities. C5a is believed to play an important role in the pathophysiology of certain skin disorders and systemic diseases with cutaneous manifestations. However, there is very little known about the in vivo reactivity of C5a in man. In this study we examined the effects of intradermal injections of human C5a in 17 normal volunteers. C5a was prepared by interacting highly purified human C5 with zymosan bound alternative pathway C5 convertase under conditions resulting in consumption of approximately 90% of the C5 substrate. C5a produced in this manner was chemotactic for human neutrophils and monocytes (0.5 X 10(-7) to 10(-9) M) and caused neutrophil aggregation and myeloperoxidase release (concentrations greater than or equal to 10(-10) M) in vitro. In vivo, C5a produced immediate wheal and flare reactions in all volunteers, and was active in doses as low as 1 ng (10(-13) mol). Intradermal testing with 20 ng of C5a in eight volunteers produced a maximal mean wheal of 11.75 mm (+/- 0.80 mm SEM) 20 min after anaphylatoxin injection, and a maximal mean erythema of 62.50 mm (+/- 3.27 mm SEM) 10 min after C5a administration. Reactions at C5a test sites were dose-related, associated with marked pruritus in some subjects, resolved without lesion formation, and were not associated with late phase reactions. In vivo testing revealed that human C5a was a more potent mediator of wheal and flare reactions than histamine, 48/80, human C3a, or morphine sulfate. Skin biopsies from eight volunteers 20 min after intradermal injection of 20 ng of C5a revealed a neutrophil-predominant perivascular infiltrate, endothelial cell edema, and sites of leukocytoclasis. Mast cell degranulation was observed on both light and electron microscopy of biopsies from C5a test sites. Although erythema at C5a injection sites was reduced by pretreating volunteers with hydroxyzine, whealing reactions and cellular infiltrates in biopsies were unaffected by this H1-antihistamine. Moderate doses of systemic corticosteroids did not alter clinical or histologic reactions at C5a injection sites in two volunteers. This study, using doses within the potential physiologic range of the anaphylatoxin, provides a comprehensive assessment of the effect of human C5a on normal human skin.

Adult↗
PubMed1979

Generation of biologic activity from the purified alpha-chain of C5.

The alpha- and beta-chains of the fifth component of human complement (C5) have been isolated and their physical and biologic properties have been characterized. After electrophoresis in SDS-poly-acrylamide slab gels, the chains were eluted from the gels and extensively dialyzed. The amino acid composition of each chain was then determined. On SDS-gels, the alpha-chain was PAS positive whereas the beta-chain revealed little or no staining. As a measure of chemotactic activity, the lysosomal enzyme-releasing activity of both chains was examined before and after trypsinization; activity resided almost entirely in the alpha subunit. Reconstitution of C5 from its isolated chains, followed by trypsinization resulted in slightly less activity than that from the individual alpha subunit. These findings provide direct support for previous evidence suggesting the biologic activity for neutrophils derives entirely from the alpha subunit of C5. This represents the first report of direct recovery of biologic activity from a purified subunit chain of C5.

Alkylation↗
PubMed1977

Hereditary C5 deficiency in man. III. Studies of hemostasis and platelet responses to zymosan.

Platelet-rich-plasma from two hemostatically normal individuals, genetically lacking the fifth component of complement (C5), failed to exhibit normal platelet aggregation, or serotonin release, in the presence of zymosan. This abnormality was found to reside in the C5D plasma rather than in the platelets as demonstrated by the inability of the deficient plasma to activate zymosan for the aggregation of washed normal platelets. The defect could be corrected by the addition of normal plasma, normal serlm, or highly purified human C5. A plasma abnormality similar to that found in the C5D individuals was also noted in plasmas deficient in C3, C6, and C7; whereas C8 plasma D behaved normally. These data suggest that this platelet reaction requires late acting C components, perhaps as the C567 complex, bound to the zymosan particles.

Blood Platelets↗
PubMed1987

Complement activity in normal rabbit bronchoalveolar fluid. Description of an inhibitor of C3 activation.

Hemolytically active C4, C3, C5, and C6, and trace amounts of C1, were present in bronchoalveolar lavage fluid from healthy adult rabbits. Both the alternative and classical pathways were functionally intact through C5. Complement depletion by intravascularly administered cobra venom factor caused a parallel decrease in lavageable C5 from the rabbit's lungs. Whereas BALF C5 had specific activity comparable to serum C5, that of BALF C3 was lower than predicted. This was found to be due to the action of a substance that selectively blocked C3 activation. The inhibitor did not cause irreversible inactivation of C3, and it had no effect on C5 activity. It blocked the formation of EAC423 from EAC42 + C3, but not the lysis of preformed EAC423 + C5-9 or EAC423 rosetting with rabbit neutrophils. Hydrophobic chromatography of BALF separated the activity of the inhibitor from that of C3. Further chromatography showed that it had a net negative charge at physiologic pH, and molecular weight between 14 and 30 kilodaltons. The presence of an inhibitor of C3 activation in the airways would provide control of complement activation in an environment that is constantly exposed to bacteria, dust, and other potential inflammatory stimuli.

Animals↗
PubMed1976

Hereditary deficiency of the fifth component of complement in man. I. Clinical, immunochemical, and family studies.

The first recognized human kindred with hereditary deficiency of the fifth component of complement (C5) is described. The proband, a 20-year-old black female with systemic lupus erythematosus since age 11, lacked serum hemolytic complement activity, even during remission. C5 was undetectable in her serum by both immunodiffusion and hemolytic assays. Other complement components were normal during remission of lupus, but C1, C4, C2, and C3 levels fell during exacerbations. A younger half-sister, who had no underlying disease, was also found to lack immunochemically detectable C5. By hemolytic assay, she exhibited 1-2% of the normal serum C5 level and normal concentrations of other complement components. C5 levels of other family members were either normal or approximately half-normal, consistent with autosomal codominant inheritance of the gene determining C5 deficiency. Normal hemolytic titers were restored to both homozygous C5-deficient (C5D) sera by addition of highly purified human C5. In specific C5 titrations, however, it was noted that when limited amounts of C5 were assayed in the presence of low dilutions of either C5D serum, curving rather than linear dose-response plots were consistently obtained, suggesting some inhibitory effect. Further studies suggested that low dilutions of C5D serum contain a factor (or factors) interfering at some step in the hemolytic assay of C5, rather than a true C5 inhibitor or inactivator. Of clinical interest are (a) the documentation of membranous glomerulonephritis, vasculitis, and arthritis in an individual lacking C5 (and its biologic functions), and (b) a remarkable propensity to bacterial infections in the proband, even during periods of low-dose or alternate-day corticosteroid therapy. Other observations indicate that the C5D state is compatible with normal coagulation function and the capacity to mount a neutrophilic leukocytosis during pyogenic infection.

Adult↗
PubMed1987

Generation of a monoclonal antibody to mouse C5 application in an ELISA assay for detection of anti-C5 antibodies.

We have generated a monoclonal antibody with specificity for the fifth component of mouse complement (C5). This antibody precipitates the two chains of C5 from normal mouse serum and inhibits C5-dependent hemolysis in a functional complement test. In this study we describe its application in an enzyme-linked immunoadsorbent assay (ELISA assay) for the detection of anti-C5 antibodies in serum. Monoclonal anti-C5 coupled to wells of an ELISA plate specifically binds C5 from unfractionated normal mouse serum. This subsequently serves as antigen to bind anti-C5 serum antibodies. By this approach we have circumvented the need for extensive purification of C5 from serum which would be required if C5 was directly coupled to ELISA plates as antigen. Serum antibodies from C5-immunized mice bound with high avidity to wells containing normal serum as antigen source in amounts representing 1 microgram to 250 ng C5. There was no antibody binding to wells containing C5-deficient serum as antigen source. The immune reaction was detected by development with enzyme-coupled goat-anti mouse Ig antibodies specific for various mouse Ig subclasses. This method allows the qualitative characterization of immune responses to mouse C5 which is an ideal model for a natural self antigen in studies of immunological tolerance.

Animals↗
PubMed1977

Hereditary C5 deficiency in man: genetic linkage studies.

Genetic linkage studies were performed on the only reported kindred with genetic deficiency of the fifth component of complement (C5). Thirty family members in four generations were studied for C5 defiency and 32 genetic marker systems. Of these marker loci, 13 were informative in this pedigree. Most importantly, C5 deficiency was excluded (lod score greater than -2.0) from linkage with the major histocompatibility locus (HLA) from a recombination frequency of greater than 15% (in females). Other marker systems excluded from linkage with C5 deficiency included the ceruloplasmin and Duffy loci at a recombination frequency of less than 15%, and the erythrocyte glyoxalase, MN, and Lewis loci at a recombination frequency of less than 5%. The most positive lod score (1.07, theta=0.05) was for linkage between C5 and haptoglobin, but this score does not reach statistical significance. Thus, among the genes for complement components which can be mapped because of deficiency states or polymorphic gene products, C5 joins C1r, C3 and C6 in not being closely linked to HLA. In contrast, close HLA linkage has been demonstrated for C2, C4, properdin factor B and, in one of two families, C8.

Adolescent↗
PubMed1977

Metabolism of the fifth component of complement, and its relation to metabolism of the third component, in patients with complement activation.

The metabolism of the fifth component of complement (C5), and its relatonship to metabolism of the third component of complement (C3), has been studied in normal subjects and patients by simultaneous administration of radioiodine labeled C5 and C3. In seven normal subjects the fractional catabolic rate of C5 ranged from 1.5 to 2.1% of the plasma pool/h and extravascular/intravascular distribution ratio from 0.22 to 0.78, these values being similar to those obtained for C3, and synthesis rate from 71 to 134 mug/kg per h, In patients with complement activation the increase in fractional catabolic rate of C5 was nearly always less than that of C3. The data also showed that there was increased extravascular distribution of C3 and C5 in most patients and considerable extravascular catabolism of both proteins in some. However, there were differences in metabolic parameters between patients with different types of complement activation. In patients with systemic lupus erythematosus, fractional catabolism and extravascular distribution of C3 and C5 were both increased, and there was marked extravascular catabolism of both proteins. There was increased fractional catabolism and extravascular distribution of C3 in patients with mesangiocapillary nephritis and (or) partial lipodystrophy, and fractional catabolism of C5 was also increased in three of six studies although distribution of C5 was always within the normal range; however, in two patients with nephritic factor in their serum fractional catabolism of C5 was normal despite markedly increased C3 turnover, suggesting that in patients with alternative pathway activation by nephritic factor little or no C5 convertase is generated.

Adolescent↗