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PubMed1992

Formation and structure of the C5b-7 complex of the lytic pathway of complement.

The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.

Amino Acid Sequence↗

PubMed1975

Cytolytic lymphocytic cells with complement receptor in human blood. Induction of cytolysis by IgG antibody but not by target cell-bound C3.

Human blood lymphocytes were fractionated on glass bead columns charged with sheep erythrocyte (Es) membranes-bearing human C3b (7,000-10,000 molecules/Es). In the passaged cells the proportion of C receptor lymphocytes was strongly reduced, in parallel with the capacity to lyse chicken erythrocytes (Ec) in the presence of IgG-rabbit anti-Ec antibody. In other experiments, lymphocytes forming rosettes with Es bearing activated rabbit complement [C(ra)] from C6-deficient rabbits were removed by centrifugation through human serum albumin-gelatine mixtures. This procedure also depleted the lymphocyte preparations of antibody-dependent cytolytic effector cells. The results suggest that rations of antibody-dependent cytolytic effector cells. The result suggest that such effector cells have receptors for human C as well as for C(ra). Lymphocytes were not able to lyse erythrocytes bearing either human C3b (similar to 30,000 molecules/Ec) or activated C(ra) in the absence if IgG antierythrocyte antibodies. Under the same experimental conditions these target cells were efficiently lysed in the presence of small amounts of IgG antitarget cell antibodies. This suggests that the interaction between the cellular Fcreceptors and the Fc part of the inducing antibodies is of special significance for the triggering of the cell-mediated lytic reaction. However, although target cell-bound C did not trigger cytolysis, it seemed to potentiate antibody-dependent cytolysis, probably by enhancing effector cell-target cell contacts.

Animals↗

PubMed1989

Non-enzymic activation of the fifth component of human complement, by oxygen radicals. Some properties of the activation product, C5b-like C5.

Purified human C5 was converted non-enzymically to an activated form as defined by its ability to participate in reactive lysis. This conversion occurred following exposure to systems that generate oxygen radicals, namely addition of H2O2 in the presence of ascorbic acid and iron or the addition of xanthine oxidase, acetaldehyde and iron. The conversion of C5 to a functionally active species was iron-dependent and inhibited by hydroxyl radical scavengers such as DMSO. The findings suggest that OH. is the active oxygen species that converts C5. The conversion product of C5, termed C5(H2O2), is C5b-like due to its ability to bind C6 and cause reactive lysis. C5(H2O2) is much more stable than C5b obtained by complement convertases. Although C5(H2O2) has lost the binding site of native C5 for C3b it can be cleaved by complement-derived convertases; the cleavage is, however, less efficient than in the case of native C5. The resulting cleavage product, which is C5a-like, is chemotactic although C5(H2O2) is not chemotactic. C5(H2O2) serves as a better substrate for plasma kallikrein than native C5, resulting in the generation of a C5a-like chemotactic product. These data indicate that oxygen radicals can bring about a conformational change in C5, causing it to behave as a functionally activated molecule of the complement system. This may have implications for the role of complement and its activation in the inflammatory response.

Complement Activation↗

PubMed1985

Transmembrane channel-formation by five complement proteins.

Five serum proteins act in concert to form the membrane attack complex (MAC) of complement. The precursor proteins, C5, C6, C7, C8 and C9, are hydrophilic glycoproteins with molecular weights ranging from 70,000 to 180,000. When C5 is cleaved by the serine protease C5 convertase, nascent C5b is produced which forms together with C6 a soluble and stable bimolecular complex (C5b,6). Upon binding of C5b,6 to C7 a trimolecular complex (C5b-7) is formed, which expresses a metastable membrane binding site. Membrane-bound C5b-7 constitutes the receptor for C8 and the tetramolecular C5b-8 complex binds and polymerizes C9. During the assembly process the proteins undergo hydrophilic-amphiphilic transition and the end product consists of C5b-8 (Mr approx. 550,000) and of tubular poly C9 (Mr approx. 1,100,000). The functional channel size varies but its maximal diameter is approximately 10 nm. C9 polymerization appears to involve initial reversible associations of several C9 molecules, which leads to temperature dependent, constrained unfolding. Unfolded C9 monomers then associate laterally with each other and polymerization terminates with closure of the circular structure, which consists of 12-18 C9 monomers. Amino acid composition and sequence indicate that the N-terminal half of the single chain C9 molecule is hydrophilic and the C-terminal half rather hydrophobic. Phospholipid binding and insertion into membranes are functions of the C-terminal portion of the molecule.

Amino Acid Sequence↗

PubMed2001

C5b-9 terminal complement complex protects oligodendrocytes from death by regulating Bad through phosphatidylinositol 3-kinase/Akt pathway.

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a G(i)alpha-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.

Animals↗

PubMed1985

C6-deficiency in rabbit serum and its restoration by normal rabbit serum or by normal bovine serum.

The sera from 12 out of 29 half-sibs of the New Zealand White breed of rabbit were inactive, when used as complement, in blood-typing tests of bovine erythrocytes. This defect was found to be due to a deficiency in the activity of the component C6. A dilution of 1:380 of pooled normal rabbit sera and a dilution of 1:520 of pooled normal bovine sera were capable of restoring, to 63%, the haemolytic capacity of the defective rabbit sera. The rate of haemolysis varied from 4% to 100% when the defective sera were restored by sera from different sets of monozygous bovine twins.

Animals↗

PubMed1976

Humoral immunostimulation. VI. Increased calcium uptake by cells treated with antibody and complement.

When L cells were treated with anti-L cell antibody in medium depleted of complement, rapid increases in calcium uptake were obtained over a wide range of antiserum concentrations. Concomitant cell growth and viability studies demonstrated that stimulation of cell growth occurred at higher dilutions of antiserum whereas cytotoxicity occurred at lower dilutions. The stimulatory and toxic effects of antibody on cell growth were potentiated by complement as was the enhancement in calcium uptake. Sera deficient in C1R, C2,4D, C4, C3-C9 did not increase the calcium uptake response to antibody whereas augmentation did occur with C6-deficient serum. A specific role for complement was further indicated by the ability of purified complement components to restore the response to complement in complement-deficient sera. C3 with C3-C9 deficient serum, but not C2, C5, and C6 with C3-C9 deficient serum restored augmentation effects. Taken together with the results of previous studies it is apparent that complement augments both calcium and nucleoside uptake and that the effect is primarily via the classical complement pathway through C3. Substrate saturation studies demonstrated that antibody activated the facilitated diffusion of calcium altering the Vmax but not the Km of transport whereas addition of complement altered both the Vmax and Km. These findings suggest that one of the early effects of enhancing antibody upon tumor cell metabolism in vitro is to stimulate uptake of calcium. In view of the suspected role of Ca++ in cell proliferation the increase in cell-associated calcium may be important in the subsequent proliferative response.

Antigen-Antibody Reactions↗

PubMed1991

Complement allotypes in Japanese patients with pemphigus and bullous pemphigoid.

The allotypes of C6, C7, factor B (BF) and factor I (IF) of the human complement system were studied in 11 Japanese patients with pemphigus (5 with pemphigus vulgaris and 6 with pemphigus foliaceus) and 17 with bullous pemphigoid (BP) to investigate the genetic background of these diseases. The allotypes were detected by using isoelectric focusing and immunoblotting. The frequency for IF*A allele in the pemphigus patients was significantly higher (p = 0.009) than that in healthy controls (n = 60). A significant association of IF A allotype with pemphigus was also observed (p = 0.027), with a relative risk of 6.3. There was no association between the C6, C7, BF or IF allotypes and BP. These data suggest that IF A allotype may be an etiological genetic factor in the development of pemphigus.

Alleles↗

PubMed1991

The properdin-like type I repeats of human thrombospondin contain a cell attachment site.

Thrombospondin (TS) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to TS. These include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an RGDA sequence of TS. We have characterized a mAb against human TS, designated A4.1, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for A4.1 lies within the amino terminal half of the central stalklike region of TS which is distinct from the three known cell attachment sites. This region of TS is recovered in a 50-kD peptide after chymotryptic digestion of TS in EDTA. It contains the procollagen-like domain of TS as well as three type I repeats of a 60-residue segment homologous to two malarial proteins and the complement proteins properdin, and factors C6 through C9. The purified chymotryptic fragment is an effective attachment factor for G361 cells. A4.1 blocks adhesion to the 50-kD domain, as do some sulfated glycoconjugates. RGD (and RGE) peptides and mAbs against other domains of TS are not inhibitory. Peptides (19 mers) based on the core homology sequence of the three type I repeats of TS are potent attachment factors for these cells, and this adhesion is also inhibited by sulfated glycoconjugates. A polyclonal antibody raised against one of these peptides inhibits adhesion of G361 cells to the peptides, to the 50-kD fragment and to intact TS. Thus a new cell-adhesion site has been identified in TS whose sequence is very similar to the site identified in region II of the circumsporozoite protein of malaria parasites (Rich, K. A., F. W. George IV, J. L. Law, and W. J. Martin. 1990. Science (Wash. DC) 249:1574-1577. Thus there may be a common receptor which binds TS, malarial proteins, and properdin.

Amino Acid Sequence↗

PubMed1990

Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression.

We have isolated the mouse thrombospondin (TS) gene and determined the DNA sequence of the first nine exons and eight introns. Comparison with the human cDNA sequence reveals a high degree of conservation in coding sequences. Exon 3 of the mouse gene, which encodes the heparin-binding domain of TS, has a higher degree of nucleotide substitution than the other exons, but the distribution of charged and hydrophobic amino acids found in the human protein is generally conserved. DNA and protein sequences in exons 6-9, which encode a procollagen homology and motifs very similar to those found in at least two malarial parasite proteins, are highly conserved. The first two of the three malarial homologies in TS, which are also found in properdin and in components C6-9 of the lytic complement complex, are each encoded by a separate exon (8 and 9) in the mouse gene. Since the sequence data did not reveal substantial similarity in sequence between intron I in the human and mouse genes, we have reexamined the role of the first intron in the transcriptional regulation of the human TS gene. In accord with published studies (Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222-11227), we find that deletion of some intronic segments from TS-chloramphenicol acetyltransferase (CAT) constructs reduces CAT activity in NIH 3T3 cells. However, deletion of the same sequences from TS-bovine growth hormone constructs does not affect the expression of bovine growth hormone in these cells. We conclude that differences in the activity of TS-CAT constructs reflect post-transcriptional differences that are peculiar to the resulting chimeric transcripts and that there is currently no evidence for a transcriptional enhancer in the first intron of the human TS gene.

Amino Acid Sequence↗

PubMed1980

Recurrent bacterial meningitis in patients with genetic defects of terminal complement components.

Isolated genetic deficiencies of complement components in man are rare. We describe two kindreds with inborn deficiencies of either C5 or C6 in which both propositi presented with recurrent bacterial meningitis. Neisseria meningitidis was isolated from the cerebrospinal fluid of the C5-deficient patient and bactericidal activity against his autologous meningococcus was absent from whole fresh patients' serum despite a rising titre of complement-fixing antibody. The stimulated movement of normal leucocytes was impaired in the presence of C5-deficient serum but not in the presence of C6-deficient serum; neither deficiency reduced significantly the complement-dependent opsonization of Saccharomyces cerevisiae. HLA typing and complement component phenotyping showed no segregation with the complement defect in either the C5- or C6-deficient families. Normal individuals and apparent heterozygotes with approximately half the normal levels of the relevant component were found in both families, in keeping with an autosomal codominant inheritance of the defects.

Adult↗

PubMed1983

Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.

Our recent observations of a complement-mediated, cell-independent mechanism of altered glomerular permeability in rat membranous nephropathy suggested a possible role for the terminal complement pathway in the mediation of proteinuria in certain forms of glomerular disease. To directly determine whether the membranolytic terminal complement components (C5b-C9) are involved in glomerular injury, we studied the development of proteinuria in normal and C6-deficient (C6D) rabbits, in both of which a membranous nephropathy-like lesion develops early in the course of immunization with cationized bovine serum albumin (cBSA) (pI 8.9-9.2). C6 hemolytic activity of C6D was 0.01% that of control rabbits. After 1 wk of daily intravenous injections of cBSA, proteinuria developed in 71% of controls (median 154, range 1-3,010 mg/24 h, n = 24), whereas none of C6D were proteinuric (median 6, range 2-12 mg/24 h, n = 12, P less than 0.01). After 1 wk of cBSA, both groups had qualitatively identical glomerular deposits of BSA, rabbit IgG, and C3 on immunofluorescence microscopy, predominantly subepithelial electron-dense deposits on electron microscopy, and minimal glomerular inflammatory cell infiltration of glomeruli. Glomeruli were isolated from individual animals after 1 wk of cBSA and deposits of rabbit IgG antibody were quantitated by a standardized in vitro assay using anti-rabbit IgG-125I. Rabbit IgG deposits were found to be similar in control (29.8 +/- 13.2, range 12.7-48.6 micrograms anti-IgG/2,000 glomeruli, n = 6) and C6D rabbits (32.6 +/- 13.8, range 16.8-48.8 micrograms anti-IgG/2,000 glomeruli, n = 5, P greater than 0.05). After 2 wk, coincident with a prominent influx of mononuclear cells and neutrophils, proteinuria developed in C6D rabbits. These results document, for the first time, a requirement for a terminal complement component in the development of immunologic glomerular injury. Since the only known action of C6 is in the assembly of the membrane attack complex, these observations suggest that the membranolytic properties of complement may contribute to glomerular damage.

Animals↗

PubMed1979

Human complement in thrombin-mediated platelet function: uptake of the C5b-9 complex.

Thrombin-mediated platelet aggregation and release is enhanced by the presence of C3, C5, C6, C7, C8, and C9 of human complement. The interaction of thrombin with its receptor on the platelet membrane initiates activation of complement on the platelet surface. Trypsin-mediated platelet function is not enhanced by the addition of complement, probably because trypsin has no receptor on the platelet surface so activation of complement is triggered in the fluid phase and not on the platelet surface. Activation of complement by thrombin led to production of dimers of the C5b-9 complex on the platelet surface. These complexes were eluted from the platelet membrane and were identified physicochemically and morphologically. The mechanism of complement-induced enhancement of platelet function is not clear, however, it probably is mediated via the arachidonic acid transormation pathway because this activity was blocked by known inhibitors of cyclo-oxygenase, namely, aspirin and indomethacin.

Antithrombin III↗

PubMed1994

Oxidants generated by the myeloperoxidase-halide system activate the fifth component of human complement, C5.

Hypochlorite and taurine chloramine (T-NCI) convert native fifth component of human complement (C5) to an activated state. This is evident from loss of functional properties of native C5 and acquisition of a binding site for C6 which is characteristic of C5b, the physiological activation fragment of C5. The complex of activated C5 with C6 is capable of combining with the components C7, C8, and C9 forming the cytotoxic terminal complement complex C5-9. The activation of C5 and its assembly with the late reacting complement components has been detected by reactive lysis, i.e. hemolysis of unsensitized red cells upon incubation with the activated C5 and the reacting complement components C6-C9. T-NCl does, however, not cleave any peptide bond in C5 as happens in the physiological activation process but converts the intact protein to the activated form. The conversion is accompanied and probably caused by oxidation of methionine residues in the C5 protein to methionine sulfoxide. Since hypochlorite and T-NCl are biological products generated by the myeloperoxidase-halide system of stimulated leukocytes, the activation of C5 by these agents may be one way to complement activation during inflammation and tissue injury.

Complement Activation↗

PubMed1979

Neisseria meningitidis bacteremia in association with deficiency of the sixth component of complement.

The serum of a 26-year-old black man with a recent episode of meningococcemia complicated by meningitis and arthritis was found to lack hemolytic complement activity. The sixth component of complement was not detected by functional or immunochemical assays whereas other components were normal by hemolytic assay. His fresh acute-phase serum lacked complement-mediated bactericidal activity against the homologous strain of Neisseria meningitidis, but the addition of fresh normal serum or purified C6 restored bactericidal activity as well as hemolytic activity. The absence of C6 activity could not be accounted for on the basis of an inhibitor. Opsonization and chemotaxis functioned normally. Histocompatibility typing of family members did not demonstrate evidence for genetic linkage of C6 deficiency with the major histocompatibility loci. This report represents the first published case of C6 deficiency associated with bacteremic Neisseria infections in which antimeningococcal bactericidal antibodies have been definitively demonstrated against the homologous strain in the acute phase of the illness.

Adult↗

PubMed1989

Depletion of C6 prevents development of proteinuria in experimental membranous nephropathy in rats.

To study the possible role of the complement membrane attack complex, C5b-9, in an experimental rat model that is morphologically indistinguishable from membranous nephropathy in man (passive Heymann nephritis [PHN]), an antibody to rat C6 was used to deplete C6 levels to less than 5% of pretreatment values (C6D) during disease development. C3, C7, C8, and C9 levels were not different in C6D and control rats. After injection of nephritogenic quantities of 125I-anti-Fx1A antibody, the kinetics of disappearance of labeled IgG from the blood were identical in the complement deficient and sufficient groups, and glomerular deposition of 125I-antibody was the same in both groups at 5 days. Glomerular deposits of sheep IgG and C3 were also similar in C6D and controls, but glomerular deposits of C6 and C5b-9 neoantigens were markedly reduced or absent in C6 depleted rats. However, despite equivalent antibody deposits, proteinuria was abolished in C6D rats compared with normocomplementemic controls. Similar results were obtained when F(ab')2 anti-rat C6 IgG was used to deplete C6 during development of PHN. These results demonstrate that C6 is required for the development of the increased glomerular permeability that occurs in PHN, presumably because C6 is required for formation of C5b-9. We conclude that glomerular injury in the PHN model of membranous nephropathy in the rat is mediated by C5b-9.

Animals↗

PubMed2026

Normoalbuminuric and albuminuric diabetic kidney disease exhibit divergent renal proteomic characteristics: implications for management.

BACKGROUND: The pathogenesis of diabetic kidney disease (DKD) is complex. Normoalbuminuric diabetic kidney disease (NADKD) is a special subtype of DKD that often progresses insidiously without detectable albuminuria, posing diagnostic and therapeutic challenges. Its pathogenesis remains unclear. Proteomic analysis of renal tissues may offer insights into its pathogenesis and identify biomarkers. METHODS: Clinicopathological data from 295 biopsy-proven DKD patients were collected and classified into normoalbuminuric (UACR < 30 mg/g, n = 25), microalbuminuric (UACR 30-300 mg/g, n = 26), and macroalbuminuric (UACR > 300 mg/g, n = 244) groups. Laser microdissection combined with mass spectrometry (LMD/MS) was used to analyze glomerular and proximal tubule proteomics in 5 patients per DKD subgroup and 5 control subjects. Associations with clinical features were examined. RESULTS: Glomerular proteomic analysis revealed that oxidative stress and metabolic pathways (UQCRC1) were upregulated in NADKD group, whereas the complement and coagulation cascades (C3, C5, C6, C9, CFH, CFHR1) were significantly upregulated in the microalbuminuric and macroalbuminuric DKD groups. The proximal tubule proteomics analysis showed that oxidative phosphorylation-related proteins (SDHA, CYCS, UQCRQ) were upregulated in NADKD, and collagen I related proteins (COL1A1, COL1A2) were significantly upregulated. CONCLUSION: Oxidative stress and mitochondrial dysfunction are involved in the progression of NADKD, lesions predominantly located in the tubulointerstitium. The complement pathway participates in the pathogenesis and progression of albuminuric DKD (ADKD). These divergent molecular profiles suggest that NADKD and ADKD may reflect different pathophysiological mechanisms and have important implications for therapeutic strategies in diabetes management.

Humans↗

PubMed1996

Physical linkage and orientation of the human complement C8 alpha and C8 beta genes on chromosome 1p32.

Human C8 is one of five components (C5b, C6, C7, C8, C9) of the cytolytic C5b-9 complex of complement. It consists of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma), which are encoded in separate genes. Genetic linkage and chromosomal localization studies previously established that C8 alpha and C8 beta are closely linked on chromosome 1p32. In this study, clones with inserts containing genes for both C8 alpha and C8 beta were isolated from a yeast artificial chromosome (YAC) human genomic DNA library and characterized in an effort to determine intergenic distance and orientation. One clone with a approximately 330-kb insert yielded restriction digest patterns for C8 alpha and C8 beta that agreed with those obtained previously from digests of human genomic DNA, thereby confirming the presence of intact copies of both genes. A second clone with a approximately 280-kb insert yielded similar results; however, it was truncated at the 5' end of the C8 alpha gene. Restriction digests of both clones were subjected to PFGE and Southern blot analysis using probes specific for the terminal exons of C8 alpha (exons 1 and 11) and C8 beta (exons 1 and 12). Results indicate the genes are physically linked (< 23 kb) and in a 3'-3' orientation. This is the same orientation as the ancestrally related C6 and C7 genes, which are also physically linked on chromosome 5p13.

Chromosome Mapping↗