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At least 19 recordsLinked to original sources
PubMed1995

Identity of the segment of human complement C8 recognized by complement regulatory protein CD59.

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Amino Acid Sequence↗
PubMed2002

Interaction between the C8 alpha-gamma and C8 beta subunits of human complement C8: role of the C8 beta N-terminal thrombospondin type 1 module and membrane attack complex/perforin domain.

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex (MAC). It is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain an N-terminal thrombospondin type 1 (TSP1) module, a low-density lipoprotein receptor class A (LDLRA) module, an extended central segment referred to as the membrane attack/perforin (MACPF) domain, an epidermal growth factor (EGF) module, and a second TSP1 module at the C-terminus. In this study, the segment of C8beta that confers binding specificity toward C8alpha-gamma was identified using recombinant C8beta constructs in which the N- and/or C-terminal modules were deleted or exchanged with those from C8alpha. Constructs were tested for their ability to bind C8alpha-gamma in solution and express C8 hemolytic activity. Binding to C8alpha-gamma was found to be dependent on the TSP1 + LDLRA + MACPF segment of C8beta. Within this segment, the TSP1 module and MACPF domain are principally involved and act cooperatively to mediate binding. Results from activity assays suggest that residues within this segment also mediate binding and incorporation of C8 into the MAC.

Amino Acid Motifs↗
PubMed2000

An indel within the C8 alpha subunit of human complement C8 mediates intracellular binding of C8 gamma and formation of C8 alpha-gamma.

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.

Amino Acid Sequence↗
PubMed1988

Sequence homology of complement C8 gamma chain with alpha 1-microglobulin and its implications for C8 structure and function.

Complement C8 gamma is a disulphide bonded subunit of C8 with no known homology or function. We show here that it strongly resembles alpha 1-microglobulin and protein HC in both length and sequence suggesting a common genetic origin. By extrapolation from the alpha 1-microglobulin family we are able to suggest that the cysteine residue of C8 gamma is involved in disulphide bonding to C8 alpha, and a possible physiological role in the regulation of the inflammatory response. Comparison of C8 gamma with C8 alpha shows that one region of C8 alpha bears a structural homology with the region of C8 gamma containing the free cysteine residue. This homology includes a cysteine residue of C8 alpha, for which no equivalent is found in C8 beta or C9, and which is therefore a likely candidate for C8 gamma attachment.

Alpha-Globulins↗
PubMed1986

The terminal complement complex in sera deficient in the eighth component of complement (C8).

The terminal complement complex (TCC) was quantified in sera from patients with a genetic deficiency of C8 alpha-gamma or C8 beta. The individual sera contained only trace amounts of TCC compared with a normal serum pool. The content of TCC increased after mixing the two sera, which was consistent with reconstitution of C8 activity. Only a moderate increase in TCC was obtained after zymosan activation of the individual sera, whereas activation of the mixture resulted in high amounts of TCC. C8 was demonstrated in the TCC of both deficient sera. These results may indicate that functional C8 is present in trace amounts despite the genetic deficiency, and that the terminal pathway may function to some extent although not enough to be detectable in less sensitive assays.

Antibodies, Monoclonal↗
PubMed1983

Genetic polymorphism in C8 beta-chains. Evidence for two unlinked genetic loci for the eighth component of human complement (C8).

Genetic polymorphism in the beta-subunit of the eighth component of human complement, C8, was defined by isoelectric focusing of serum in polyacrylamide gel in the presence of urea and development of specific patterns of hemolysis in an overlay gel containing antibody-sensitized erythrocytes and C8 beta-chain-deficient serum. Bands of hemolysis induced by serum from unrelated Caucasians suggested autosomal codominant inheritance of three structural alleles at a single locus, C82: C82 degrees A (acidic), C82 degrees B (basic), and C82 degrees A1 (very acidic) with frequencies of 0.952, 0.044, and 0.004, as well as the probable null allele C82 degrees Q0. The distribution of phenotypes agreed with the Hardy-Weinberg equilibrium. The previously described genetic polymorphism in human C8 defined with the use of "complete" C8 (C8 alpha-gamma-chain)-deficient serum was distinct from and independent of the inherited structural variation at C82. Therefore, the locus for C8 alpha-gamma-chains has been redesignated C81, and has the alleles C81 degrees A, C81 degrees A1, and C81 Q0. Linkage studies failed to show close linkage between the two loci for C8, C81, and C82, and between C82 and the major histocompatibility complex or C6.

Alleles↗
PubMed1993

Genetic basis of human complement C8 beta deficiency.

The eighth component of human complement (C8) is a serum protein consisting of three chains (alpha, beta, and gamma) and encoded by three different genes, C8A, C8B, and C8G. C8A and C8B are closely linked on chromosome 1p, whereas C8G is located on chromosome 9q. In the serum the beta subunit is non-covalently bound to the disulfide-linked alpha-gamma subunit. Patients with C8 beta deficiency suffer from recurrent neisserial infections such as meningitis. Exon-specific polymerase chain reaction (PCR) amplification with primer pairs from the flanking intron sequences was used to amplify all 12 C8B exons separately. No difference regarding the exon sizes was observed in a C8 beta-deficient patient compared with a normal person. Therefore, direct sequence analysis of all exon-specific PCR products from normal and C8 beta-deficient individuals was carried out. As a cause for C8 beta deficiency, we found a single C-T exchange in exon 9 leading to a stop codon. An allele-specific PCR system was designed to detect the normal and the deficiency allele simultaneously. Using this approach as well as PCR typing of the Taql polymorphism located in intron 11, five families with 7 C8 beta-deficient members were investigated. The mutation was not found to be restricted to one of the two Taql RFLP alleles. The mutant allele was observed in all families investigated and can therefore be regarded as a major cause of C8 beta deficiency in the Caucasian population. In addition, two C8 beta-deficient patients were found to be heterozygous for the C-T exchange. The molecular basis of the alleles without this point mutation also causing deficiency has not yet been defined.

Base Sequence↗
PubMed1997

Expression of the human complement C8 subunits is independently regulated by interleukin 1 beta, interleukin 6, and interferon gamma.

The eighth component of human complement (C8) is composed of two subunits which are products from three separate genes. The alpha-gamma- and the beta-subunit of C8 are expressed independently, and are part of the membrane attack complex. C8 is primarily synthesized in the liver. It has been shown in previous studies that the human hepatoma cell line HepG2 constitutively expresses C8, and thus is a suitable model system for studying C8 biosynthesis in vitro. Expression is modulated by the cytokines IL-1 beta, IL-6 and IFN-gamma. The effect of the different cytokines on the expression of these subunits was examined using biosynthetical labelling and immunoprecipitation methods. C8 alpha-gamma is expressed first and secreted independently from C8 beta. After 5 h labelling, the expression is strongly reduced, and the majority of C8 alpha-gamma is found in the supernatant. C8 beta expression exhibits a different pattern with a much slower rate of biosynthesis and secretion. Evidence was obtained for an independent secretion of the C8 beta chain. C8 alpha-gamma expression is strongly enhanced after stimulation with the cytokines IL-6, IFN-gamma and IL-1 beta. In contrast, only IFN-gamma but not IL-6 and IL-1 beta had an increasing effect on the expression of C8 beta. Thus the total amount of assembled functionally active C8 appears to be limited by the rate of C8 beta expression.

Antineoplastic Agents↗
PubMed1983

Two types of dysfunctional eighth component of complement (C8) molecules in C8 deficiency in man. Reconstitution of normal C8 from the mixture of two abnormal C8 molecules.

Restoration of hemolytic activity was examined in sera from seven unrelated eighth component of complement (C8)-deficient subjects. The sera fell into two groups, depending on whether hemolytic activity was restored by the addition of the beta-chain (group 1) or the alpha-gamma-subunit (group 2) purified from normal human C8. Antigenic analysis of these sera by double-immunodiffusion using anti-human C8 confirmed previous findings of a dysfunctional C8 in the four sera of group 1 and established the presence of a different dysfunctional C8 in one of the sera of group 2 when tested at a high concentration. Further characterization of the dysfunctional C8 molecules in the two sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that group 1 sera were missing the beta-subunit and group 2 sera were missing the alpha-gamma-subunit of the C8 molecule. Sera from either of these two groups alone did not produce hemolysis in hemolytic plates containing sheep erythrocytes coated with antibody and complement components up to C7 (EAC1-7) and C9. When sera from the two groups were added to adjacent wells in the hemolytic plates, a zone of hemolysis developed between the wells. The contribution of C8 alpha-gamma from the sera of group 1 and of C8 beta from those of group 2 to the lysis of EAC1-7 in the presence of C9 was confirmed by the inhibitory effect of specific antibodies against the two C8 subunits. In experiments in which hemolytic activity was reconstituted by mixing sera from group 1 with sera from group 2, the serum source of C8 beta (group 2) was the limiting reagent. The dysfunctional C8 molecule in this serum was able to bind to EAC1-7. Chromatographic analysis demonstrated that the generation of hemolytic activity in the mixture of the two sera resulted from the reconstitution of the C8 molecule rather than the sequential action of the two C8 subunits.

Antibody Formation↗
PubMed1984

Recurrent Neisseria meningitidis bacteremia. Association with deficiency of the eighth component of complement (C8) in a Sephardic Jewish family.

A 24-year-old man had repeated episodes of meningococcal meningitis. Selective deficiency of the eighth component of complement (C8) was demonstrated in the patient, his twin brother, and in one of five siblings. As the parents were first cousins of normal phenotype, this pattern is suggestive of an autosomal recessive heredity. The present report brings the total number of patients given the diagnosis of C8 deficiency to 14, and calls attention to the existence of this condition in Jews of Sephardic (Mediterranean) origin.

Adult↗
PubMed1983

Interaction of the eighth component of guinea pig complement (C8) with the membrane-bound C5b-7 complex. The binding site of C8 to C5b-7 is formed on association of the alpha-gamma subunit with the beta-chain.

The eighth component (c8) of guinea pig complement consists of three polypeptide chains, the alpha-, beta-, and gamma-chains with M.W. of 60,000, 60,000, and 24,000, respectively. The alpha- and gamma-chains are bound by a disulfide bond(s) forming an alpha-gamma subunit, which is linked noncovalently to the beta-chain. The alpha-gamma subunit and the beta-chain were separated and purified from C8 by treatment with sodium dodecyl sulfate (SDS) and gel chromatography on Sephacryl S-300 in the presence of SDS. After removal of SDS, neither alpha-gamma nor beta showed the hemolytic activity of C8 when assayed independently, but showed significant activity in combination, indicating reconstitution of active C8. The recovery of hemolytic activity was 3.48%. When alpha-gamma and beta were incubated successively with EAC-7 with intervening reconstitution of active C8 on the cells was insignificant, irrespective of the order of the reactions. alpha-gamma and beta did not bind to EAC-7 when added separately, but after recombination 7% of alpha-gamma and 9% of beta bound to EAC-7 when EAC-7 was in excess. These results indicate that the binding site of guinea pig C8 to the membrane-bound C5b-7 complex does not exist on either alpha-gamma or beta only but stretches over both or is formed on one subunit after recombination of the subunits.

Animals↗
PubMed2006

Incorporation of human complement C8 into the membrane attack complex is mediated by a binding site located within the C8beta MACPF domain.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the membrane attack complex (MAC). C8 is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain N- and C-terminal modules and an intervening approximately 40 kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. C8beta participates in at least two binding interactions. It has a high affinity binding site for C8alpha, which facilitates its interaction with C8alpha-gamma. C8beta also mediates incorporation of C8 into the MAC by binding to C5b-7, an intermediate in the MAC assembly pathway. Little is known about the location or properties of the respective binding sites on C8beta. In this study, the MACPF domain of C8beta (betaMACPF) was expressed in Escherichia coli and its role in binding C8alpha and C5b-7 examined. Recombinant betaMACPF was shown to bind C8alpha-gamma in solution and form a noncovalent complex (betaMACPF*C8alpha-gamma) that exhibited C8 hemolytic activity. betaMACPF was also capable of binding independently to erythrocytes carrying C5b-7. Subsequent addition of C8alpha-gamma and C9 to these cells produced a hemolytically active MAC. The ability to produce a soluble, recombinant betaMACPF that retains the binding functions of C8beta suggests this segment of C8beta is an independently folded domain. Furthermore, results indicate the principal binding sites for C8alpha and C5b-7 are located within this domain, and that C8beta binding specificity is not determined by the N- and C-terminal modules.

Binding Sites↗
PubMed1969

The eighth component of human complement (C8): isolation, characterization, and hemolytic efficiency.

A method was described allowing the isolation of the eighth component of complement, C8, from human serum. C8 is an immunochemically definable, heretofore unrecognized serum protein with a molecular weight of approximately 150,000 and an electrophoretic mobility of a gamma(1)-globulin. It may be obtained with 5% yield as a 5000-fold purified, physicochemically and immunochemically homogeneous protein. Under optimal experimental conditions, a few molecules of C8 were found sufficient for lysis of a cell.

Animals↗
PubMed1994

Comparative mapping of lipocalin genes in human and mouse: the four genes for complement C8 gamma chain, prostaglandin-D-synthase, oncogene-24p3, and progestagen-associated endometrial protein map to HSA9 and MMU2.

The lipocalin superfamily encompasses a large set of quite distantly related proteins that act as carriers for small, lipophilic molecules. The lipocalin genes coding for orosomucoid, the alpha 1-microglobulin/bikunin precursor, and the major urinary protein map to MMU4, while their human counterparts map to the homologous HSA9q34 area where three other lipocalin genes for complement C8 gamma chain (C8G), progestagen-associated endometrial protein (PAEP), and prostaglandin D synthase (PTGDS) are also located. By linkage analyses in an interspecific backcross progeny in mouse, the Lcn2 gene coding for the oncogenic lipocalin 24p3, as well as the 3 lipocalin genes for C8G, PTGDS, and PAEP, have now been assigned to MMU2. The first three genes map to proximal MMU2, which is known to be homologous to HSA9q34. Paep is more distally located, which extends the number of regions with conserved syntenies between HSA9q34 and MMU2. By in situ hybridization, the human LCN2 gene maps to HSA9q34. Our data indicate that the lipocalin locus arrangement in the human/mouse ancestor is closer to that found at HSA9q than to that in the MMU genome.

Acute-Phase Proteins↗
PubMedDate not supplied

Structural and functional characterization of complement C8 gamma, a member of the lipocalin protein family.

Human complement component C8 exhibits an unusual structure in that it contains three chains, two of which, alpha and beta, display high sequence homology to other complement and CTL pore-forming proteins. The third chain, C8 gamma, is covalently linked to C8 alpha by a disulfide linkage; it is demonstrated that Cys40 of C8 gamma is linked to Cys164 of C8 alpha, a unique cysteine located in a loop located between the cysteine-rich LDL-receptor class A module and the membrane-inserting region of C8 alpha. C8 gamma was recently identified as a member of the lipocalin protein family, in which all proteins were either shown to, or are believed to bind small hydrophobic ligands. The present results now demonstrate that C8 gamma incorporates retinol and retinoic acid in the presence of 2 M NaCl. Molecular modeling of C8 gamma, based on the crystal structure of the homologous beta-lactoglobulin, reveals a structure of eight antiparallel beta-strands, bearing a highly hydrophobic binding pocket. The residues participating in the pocket formation are highly conserved when compared with the structures of beta-lactoglobulin and retinol-binding protein, both of which are known to interact with retinol. It is therefore proposed that C8 gamma may act as a retinol transporting protein in plasma.

Alpha-Globulins↗
PubMed1987

Complementary DNA cloning of complement C8 beta and its sequence homology to C9.

The complete amino acid sequence of mature C8 beta has been derived from the DNA sequence of a cDNA clone identified by expression screening of a human liver cDNA library. Comparison with the amino acid sequence of C9 shows an overall homology with few deletions and insertions. In particular, the cysteine-rich domains and membrane-inserting regions of C9 are well conserved. These findings are discussed in relation to a possible mechanism of membrane attack complex formation.

Amino Acid Sequence↗