Regional chromosomal assignment of genes encoding the alpha and beta subunits of human complement protein C8 to 1p32.
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Cells, Cultured↗
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Human complement C81 (C8 A) polymorphism: detection and segregation of new variants.
In addition to the earlier detected C81(A) rare variants A1, A2 (now A3) and B1 (now B2), six new rare variants (C81 A2 new, A4, A5, A6, M1 and B1new) are described within the polymorphism of the eighth component of human complement (alpha-gamma chain subunit). Except for A3, all rare C81 A variants are only detected by isoelectric focusing, and not by SDS polyacrylamide gel electrophoresis (PAGE), in the alpha-gamma subunit. In one individual out of approximately 700 individuals studied, a reversed position of the common allele (B vs A) was observed by SDS PAGE and the isofocusing technique. The segregation of A1, A3 and A4 could be followed in putative father/child combinations.
Alleles↗
In vitro expression of the beta subunit of human complement component C8.
Human C8 is one of five components of the cytolytic C5b-9 complex of complement. It is an oligomeric protein composed of three subunits (alpha, beta, gamma) encoded in separate genes. These are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. Biosynthesis studies and analyses of humans with hereditary C8 deficiencies suggest that C8 alpha-gamma synthesis and secretion can occur independently of C8 beta, but that serum levels of C8 beta are dependent on C8 alpha-gamma. One aim of the present study was to determine if functional human C8 beta could be synthesized in the absence of C8 alpha-gamma. Human C8 beta expression constructs were prepared and used to produce recombinant C8 beta (rC8 beta) in insect and COS-7 cells. Both cell types secreted rC8 beta that was similar in size to human C8 beta and exhibited similar ability to associate with human C8 alpha-gamma and form functional C8. A mutant form of C8 beta in which N-glycosylation sites were eliminated was also expressed and found to be functionally similar to rC8 beta and human C8 beta. These results indicate that C8 alpha-gamma is not required for intracellular processing and secretion of C8 beta. Furthermore, N-linked carbohydrate on C8 beta is not necessary for association with C8 alpha-gamma or for C8 activity.
Animals↗
Structural homology of human complement component C8 gamma and plasma protein HC: identity of the cysteine bond pattern.
Anti-C8 alpha-gamma specific antibodies were used to isolate cDNA clones from a human liver expression library. Antibodies affinity-purified on the expressed hybrid protein of one clone bound exclusively to the gamma-chain of reduced C8 alpha-gamma. This clone, as well as a second full length cDNA clone obtained by hybridization screening, were sequenced and the complete primary structure for C8 gamma was established. Cyanogen bromide cleavage of C8 alpha-gamma released a 12 kDa carboxy-terminal C8 gamma fragment under both reducing and nonreducing conditions which was identified by fragment-specific, affinity-purified antibodies. Our data clearly show that C8 gamma has one internal disulfide bridge between cys-76 and cys-168 within the carboxy-terminal 12 kDa fragment, whereas the remaining cysteine residue 40 forms the disulfide bridge with C8 alpha. The overall sequence homology to plasma protein HC (23% amino acid identities) and the conservation of one internal cysteine bond and one free, surface-located cysteine residue suggests a highly conserved three-dimensional structure of C8 gamma and protein HC and also a possible functional relationship between these proteins.
Alpha-Globulins↗
Genetic deficiency of complement component C8 in the rabbit: evidence of a translational defect in expression of the alpha-gamma subunit.
We examined the molecular basis for rabbit C8 alpha-gamma deficiency (C8D) using human C8 cDNA probes. Sequential probing of normal rabbit poly(A)+RNA revealed messages of 2.1, 1.9, and 0.8 kb for alpha, beta, and gamma, respectively. Corresponding analysis of C8D rabbit poly(A)+RNA identified messages for alpha and gamma of the same size and amounts as normal rabbits. Thus, C8 alpha-gamma deficiency in the rabbit appears to be the result of a translational rather than a transcriptional defect.
Animals↗
Expression and characterization of recombinant subunits of human complement component C8: further analysis of the function of C8 alpha and C8 gamma.
Human C8 is composed of three nonidentical subunits (C8 alpha, C8 beta, and C8 gamma) that are encoded in separate genes. In C8 isolated from serum, these are arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. In this study, a recombinant form of C8 alpha-gamma was expressed independently of C8 beta in insect cells and COS-7 cells and was shown to be equivalent to serum-derived C8 alpha-gamma with respect to its ability to combine with C8 beta and form functional C8. Also expressed separately were mutant (mut) forms of C8 alpha and C8 gamma in which the single interchain disulfide bond was eliminated. MutC8 alpha exhibited the ability to combine with C8 beta and express hemolytic activity, although at a lower level than human C8. Addition of purified mutC8 gamma increased this activity, presumably by binding to mutC8 alpha. A possible role for C8 gamma as a retinol binding protein was also investigated. Absorbance spectroscopy and fluorescence emission and quenching revealed no specific binding of retinol to mutC8 gamma. Together, these results indicate that 1) the biosynthesis and secretion of C8 alpha-gamma is not dependent on C8 beta, which is consistent with in vivo observations in C8 beta-deficient humans; 2) C8 alpha can be synthesized independently of C8 gamma; therefore, protection of C8 alpha from premature membrane interactions during biosynthetic processing is not a likely function of C8 gamma; 3) C8 gamma enhances but is not required for expression of C8 activity; and 4) C8 gamma does not bind retinol; therefore, it cannot function as a retinol transport protein.
Amino Acid Substitution↗
Genetic aspects of complement component C8 in Norwegian meningococcal disease patients.
Sera from 85 consecutive systemic meningococcal disease patients and 203 matched control individuals were C8 typed. In the patient group, one C8B deficient individual was discovered; none in the control group. No case of C8A deficiency was encountered. The material was collected during a period of epidemic meningococcal disease in Norway, mainly due to group B organisms. C8A and C8B phenotype distributions were not significantly different in the two groups. This indicates that no particular C8 type (apart from deficiency) predisposes for meningococcal disease. Neither is there any evidence of over-representation of heterozygous deficiency among meningitis patients. The C8B deficient individual and his family were studied. Tests for haemolytic complement were normal in all members except for the proband. Electrophoretic C8 patterns seemed to be slightly weaker in the heterozygously C8B deficient individuals than in persons with 2 normal C8B genes. DNA from the family members were studied with regard to a restriction fragment length polymorphism (RFLP) for the C8B gene. All exhibited the same pattern, indicating that the C8B deficiency is not due to a major deletion in the C8B gene.
Complement C8↗
Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis.
The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis.
Complement C8↗
Functional domains of the alpha subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the gamma chain.
The purified gamma subunit of the eighth component of human complement (C8) was used to characterize its site of interaction within C8 and to probe the ultrastructure of membrane-bound C5b-8 and C5b-9 complexes. Purification of gamma was accomplished by separating the disulfide-linked alpha-gamma subunit from the noncovalently associated beta chain and subjecting the former to limited reduction, alkylation, and ion-exchange chromatography. Upon mixing, purified alpha and gamma exhibited a high affinity for each other, as evidenced by their ability to form a noncovalent, equimolar complex at dilute concentrations and in the presence of excess serum albumin. Purified gamma also exhibited an affinity for C8', a previously described derivative that is functionally similar to C8 although it is composed of only alpha and beta. These results indicate that alpha possesses a specific site for interaction with gamma and that this site is preserved in the isolated subunit. Furthermore, this site remains accessible when alpha is associated with beta. In related experiments, gamma was found to specifically associate with membrane-bound C5b-8' and C5b-(8')9 complexes. These results indicate that the site for gamma interaction remains accessible on alpha in C5b-8' and is not shielded by C9 within C5b-(8')9. It is concluded that the gamma subunit of C8 is located on the surface of membrane-bound C5b-8 and C5b-9.
Binding Sites↗
Decreased C5b67-inhibitor activity in two families with hereditary functional deficiency of the eighth component of complement.
In addition to its role in hemolysis and host defense against Neisseria infection, the eighth component of human complement (C8) is one of several plasma proteins that are C5b67-inhibitors (C5b67-INH). The recent identification in our laboratory of two new families with hereditary deficiency of C8 provided an opportunity to study further the role of C8 as a C5b67-INH. Based on mixing and reconstitution experiments, the deficiency of C8 seemed to be due to a selective lack of the C8 beta-chain in one family and the C8 alpha-gamma subunit in the other family. Sera from individuals homozygous for the C8 abnormality were substantially deficient in C5b67-INH activity as well as totally deficient in hemolytic activity. Sera from control individuals possessed approximately 2500 C5b67-INH U/ml, whereas sera from the C8-deficient individuals had markedly depressed C5b67-INH activity, with a mean of only 428 U/ml. C5b67-INH activity was completely reconstituted in C8-deficient serum by the addition of purified human C8. We conclude that C8 constitutes the substantial majority of the C5b67-INH activity of normal human serum.
Adolescent↗
Regulation of complement functional efficiency by histidine-rich glycoprotein.
The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.
Animals↗
Monoclonal antibodies directed against human C5 and C8 block complement-mediated damage of xenogeneic cells and organs.
The hyperacute rejection (HAR) of xenotransplanted organs is initiated by the deposition of natural antibodies on donor endothelium followed by the activation of the recipient complement system, which rapidly destroys the graft. Studies of the role of activated complement in HAR have suggested that natural antibody as well as early (C3a, C3b) and late (C5a, C5b-9) activated complement components may contribute to cell activation and damage. Attenuation of HAR has been achieved by blockade of C3 activation with soluble CR1 or consumptive depletion of complement with cobra venom factor; however, similar studies using specific inhibitors of terminal complement components have not been described. To address the contribution of C5a and the membrane attack complex (C5b-9, MAC) to complement-mediated xenogeneic cell and organ damage, we utilized functionally blocking monoclonal antibodies directed against the human terminal complement components C5 and C8. Our data show that both anti-C5 and anti-C8 mAbs protect porcine aortic endothelial cells from membrane damage mediated by human C5b-9. Additionally, both the anti-C5 and anti-C8 mAbs blocked complement-mediated generation of membrane prothrombinase activity on porcine aortic endothelial cells challenged with human serum. To test the ability of these antibodies to attenuate antibody and complement-mediated damage of xenogeneic organs, an ex vivo model was developed wherein isolated rat hearts were perfused with human serum in the presence or absence of the anti-C5 and anti-C8 mAbs. Our data demonstrate that mAbs directed against human C5 and C8 prevented organ damage by human serum complement and suggest that these molecules may serve as potent inhibitors of HAR.
Animals↗
Role of the beta subunit in interaction of the eighth component of human complement with the membrane-bound cytolytic complex.
This study is concerned with the subunits of the eighth component of human complement (C8) and their role in facilitating specific incorporation of C8 into the membrane-bound cytolytic complex of complement. The noncovalently associated alpha-gamma and beta subunits of C8 were purified and examined for their ability to interact independently with the C8 binding site on C5b-7, the membrane-bound precursor of the cytolytic complex. Using erythrocyte-bound C5b-7 (EAC1-7), it was observed that native C8 and its isolated beta subunit have similar high affinities for this complex. Binding of beta to EAC1-7 was specific for the C8 binding site as evidenced by the fact that 1) nearly identical molar amounts of either C8 or beta were required to saturate C8 binding sites on EAC1-7; 2) pretreatment of EAC1-7 with saturating amounts of beta prevented binding of C8; and 3) pretreatment of EAC1-7 with saturating amounts of C8 prevented binding of beta. In related experiments, alpha-gamma alone had no affinity for EAC1-7, however, binding comparable to that exhibited by native C8 occurred if alpha-gamma was first incubated with an equimolar amount of beta. Other experiments showed that if EAC1-7 was first saturated with beta to produce EAC1-7(beta), alpha-gamma also bound to yield functional C8 on the cell surface. These results provide conclusive evidence that structural features of C8 which are essential for specific recognition by membrane-bound C5b-7 are contained in the beta subunit.
Cell Membrane↗
Function of subunits within the eighth component of human complement: selective removal of the gamma chain reveals it has no direct role in cytolysis.
The eighth component of human complement (C8) consists of a disulfide-linked alpha-gamma dimer that is noncovalently associated with beta. Previous results from this laboratory established that two of these subunits have distinct roles in the cytolytic function of C8. Binding of C8 to the precursive C5b-7 complex is mediated strictly through beta while interaction between C8 and the lipid bilayer of target membranes occurs primarily through alpha. In the present study, we examined the importance of the gamma subunit in cytolysis by characterizing functional properties of C8', a derivative of C8 that lacks gamma. Preparation of this derivative was accomplished by limited cleavage of disulfide bonds in purified alpha-gamma and separation of alpha from gamma. When mixed, purified alpha and beta combined to form C8'. When tested for functional similarity to normal C8, the following results were obtained. (1) Specific and saturable binding of C8' to the C8 binding site on C5b-7 could be achieved. Significantly, the resulting C5b-8' supported subsequent C9 binding and cell lysis, which was equivalent to that observed with C5b-8. (2) Complement activation of (C8 + C9)-depleted serum containing C8' resulted in formation of SC5b-8'. Inclusion of C9 in the serum resulted in further conversion to SC5b-(8')9. These observations indicate that C8' is functionally similar to C8. Furthermore, they indicate that unlike alpha and beta, the gamma subunit has no direct role in facilitating C8 interaction with the nascent cytolytic complex or in mediating C9 binding and membrane lysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression of the complement regulator factor C8 binding protein on human glomerular cells protects them from complement-mediated killing.
The complement-regulatory factor C8 binding protein (C8bp) was first identified on human erythrocyte membranes by its affinity for the complement component C8 and its ability to inhibit lysis by homologous complement. Cultured human glomerular mesangial or epithelial cells (GEC) expressed C8bp on the cell surface and in the cytoplasm. Following stimulation of the glomerular cells with interleukin 1 beta, C5b-9 or with endotoxin, a transient, protein synthesis-independent increase in C8bp surface expression was seen. Blocking of C8bp function with F(ab)2 fragment of an antibody to C8bp rendered GEC susceptible to complement-mediated killing, indicating that C8bp contributes to the cellular defense against complement attack.
Blood Proteins↗
Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8 gamma is not essential.
Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex (MAC) on bacterial cell membranes. It is an oligomeric protein composed of a disulfide-linked C8 alpha-gamma heterodimer and a non-covalently associated C8 beta chain. Previous studies revealed that C8 alpha and C8 beta have distinct roles in the formation of the MAC on simple cells such as erythrocytes and that both subunits are essential for cell lysis. These studies also determined that C8 gamma is not required for expression of MAC hemolytic activity. To determine if these conclusions are applicable to more biologically relevant systems, the C8 subunits were examined for their ability to support complement-mediated killing of Gram-negative bacteria. Results indicate: (1) C8 alpha-gamma, C8 alpha, C8 beta and C8 gamma have no independent bactericidal activity; (2) bacterial killing requires C8 beta and either C8 alpha-gamma or C8 alpha; (3) C8 alpha is an effective substitute for C8 alpha-gamma in bacterial killing; and (4) C8 gamma enhances, but is not required for C8 bactericidal activity. Together, these data suggest that C8 alpha and C8 beta have correspondingly similar roles in MAC-mediated lysis of erythrocytes and bacterial killing. Furthermore, they provide the first direct evidence that C8 gamma is not required for complement-mediated killing of Gram-negative bacteria.
Blood Bactericidal Activity↗
Relapsing Neisseria meningitidis infection associated with C8 deficiency.
Infection due to serogroup Y of Neisseria meningitidis has many clinical manifestations, ranging from mild bacteremia to fatal sepsis and meningitis. N. meningitidis infection may coincide with several complement deficiencies. A child is described who suffered from recurrent disease due to N. meningitidis, group Y, attributed to deficiency of the eighth component of complement (C8). In a review of the literature, recurrent infection occurred in six of 13 children with complement deficiency, four of whom had serotyping positive for N. meningitidis, group Y. Screening for complement deficiency is recommended for all children with meningococcal disease due to N. meningitidis, group Y, and for any child with recurrent infection due to any Neisseria species.
Complement C8↗
Antisense sequences of 20-kDa homologous restriction factor (HRF20) are found in C9 and the C8 beta chain of homologous complement.
We examined, on the basis of the fact that sense and antisense peptides have affinity for each other, whether any relationship exists between homologous restriction factor (HRF20), a membrane inhibitor of the terminal stage of homologous complement attack, and the antisense sequence of the terminal complement components C8 and C9. In this article, we demonstrate that there are two regions of C9 that contain antisense sequences to one continuous region of HRF20 and that this relationship exists between human HRF20 and human C9, but not mouse C9. We also found one region of the C8 beta chain that contains an antisense sequence to HRF20.
Amino Acid Sequence↗