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PubMed1995

Expression of the complement C8 genes during interleukin-6-mediated in vitro induction of the acute-phase response.

The three chains of the human complement component C8-alpha, beta and gamma- are encoded by distinct structural genes. C8A and C8B are closely linked on chromosome 1p and C8G is located on chromosome 9q. The biosynthesis and regulation of the gene products were studied in the human hepatoma-derived cell line HepG2 after in vitro induction of the acute-phase response by incubation with the cytokine interleukin IL-6. Analysis of C8 expression by immunoprecipitation and SDS-PAGE of biosynthetically labeled alpha-gamma and beta subunits demonstrated a positive response to this cytokine. The expression pattern observed in the hepatoma cells characterizes C8 in vitro as a positive acute-phase protein. In addition, the comparison of the relative amounts of the C8 transcripts provides evidence for a post transcriptional regulation of the C8 beta subunit. No evidence was obtained for an increased expression of IL-6 or IL-6 receptor mRNA thus excluding autoregulatory mechanisms of the cytokine in HepG2 cells.

Acute-Phase Reaction↗
PubMed1998

Genetic basis of human complement C8 alpha-gamma deficiency.

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.

Adult↗
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↗
PubMed2002

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit.

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.

Animals↗
PubMed1989

DNA polymorphism of the human complement C8 beta gene: formal genetics and intragenic localization.

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length human C8 beta cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymes Taq I and Bam HI. The Taq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are 0.68 and 0.32, respectively. The second RFLP with Bam HI is correlated with the Taq I variants: 3 kb Bam HI; 4.9 kb Taq I and 3.3 kb Bam HI; 2.8/2.1 kb Taq I. Both RFLPs could be mapped to the 3' portion of the C8 beta gene. Based on the size of genomic restriction fragments, the C8 beta gene can be estimated to have a size of 32-36 kb. Because of the even frequency distribution, the C8 beta DNA polymorphisms may be useful in gene mapping and disease association studies.

Alleles↗
PubMed2004

[Further study on heterogeneic basis of complement C8 beta deficiency].

OBJECTIVE: In Caucasian population, the most common molecular basis for C8 beta deficiency s a single C to T transition in exon 9 of C8 beta gene resulting in a stop codon. In previous family studies, two individuals were identified with C8 beta complete deficiency and were found to be only heterozygous for this mutation. This study was conducted by the present authors in search of other possible causes for these two C8 beta deficient individuals. METHODS: Using direct DNA sequence analysis of all exon-specific PCR products of the C8 beta gene from these two C8 beta deficient patients and their descendants. RESULTS: Two other C to T transitions at base 298 and 388 in exon 3 were detected, which could also create a termination codon. The descendants from one of the deficient patients were also analysed for the mutations, and it could be demonstrated that the two C to T mutations in exons 9 and 3 are segregating independently. CONCLUSION: These two mutations, which create a termination codon, are sufficient to explain the complete C8 beta deficiency in both patients.

Codon, Nonsense↗
PubMed1985

Complement C8 deficiency with recurrent meningococcemia: examination of meningococcal opsonization.

A 10 year old girl presented with recurrent febrile episodes over 2 months. A non-typable strain of Neisseria meningitidis was grown from blood cultures on three occasions. She was found to lack functional C8 activity in serum but material with C8 antigenic activity was present. The opsonic activity of the C8 deficient serum for N. meningitidis was equivalent to that of normal controls.

Child↗
PubMed1994

Homologous restriction factor: effect on complement C8 and C9 uptake and lysis.

Homologous restriction factor (HRF) is a complementary regulatory protein found on the surface of human erythrocytes and other cell types. It has the function of blocking the lytic action of the membrane attack complex (MAC) of complement when incorporated into a membrane. HRF was found to inhibit not only the MAC composed of the proteins C5b-9, but also the C5b-8 complex. When 125I-labelled HRF was allowed to insert into an erythrocyte target, lysis by C5b-8 was reduced by about 80%. When human erythrocytes were treated with antibody to HRF, which bound to and presumably blocked the action of HRF, lysis by C5b-8 increased. This increase was not caused by additional binding of C8 on the cell membrane. When HRF was inserted into a target cell membrane, it had the effect of reducing the amount of C9 that could bind to very low levels, in one experiment from 6000 molecules per cell to about 500 molecules per cell. When this value was compared to the number of C7 molecules that bound, it was found that HRF reduced the amount of C9 bound to the level of C7 that was bound. HRF seemed to reduce the number of molecules of C9 that could bind to the C5b-8 complex so that only one C9 bound per complex.

Animals↗
PubMed1984

Purification of functional subunits of the eighth component of human complement (C8) under nondenaturing conditions.

A simple procedure is described for purifying the noncovalently associated alpha-gamma and beta subunits of human C8 in the absence of denaturants and in yields significantly higher than previously obtainable. The procedure utilizes either conventional or high-pressure molecular sieve chromatography in the presence of 1.5 M NaCl. This ionic strength promotes dissociation of alpha-gamma from beta and facilitates their physical separation under mild conditions. Yields are typically 60-75% and, as judged by several criteria, each subunit is functionally identical to those isolated by earlier methods using sodium dodecyl sulfate. This new procedure eliminates the need for this detergent and thereby avoids the major solubility and yield problems encountered during its removal.

Chromatography, Gel↗
PubMed1987

Immunocytochemical localisation of complement components C8 and C9 in human diseased muscle. The role of complement in muscle fibre damage.

The localisation of the complement components C8 and C9 was studied immunocytochemically in human diseased muscle to determine the role of complement in muscle fibre damage. Monoclonal antibodies to 2 epitopes of C9 and a monoclonal antibody to the alpha subunit of C8 were applied to frozen sections of muscle biopsies from 9 cases of dermatomyositis, 5 cases of polymyositis, 7 cases of Duchenne muscular dystrophy and 4 cases of Becker muscular dystrophy. These were compared with 6 control biopsies which were morphologically normal. In all cases of inflammatory myopathies several non-necrotic fibres showed discrete peripheral patches of C9 and to a lesser extent C8. In the muscular dystrophies peripheral C9 was observed on a few non-necrotic fibres and basophilic fibres showed C9 between the fibres as well as at the periphery. In all cases necrotic fibres labelled intensely with C9 and C8 but intensities varied with the different monoclonal antibodies. This was thought to result from differences in the polymerisation of the C9 molecule in the membrane attack complex. Complement C8 and C9 were also localised to blood vessels in 3 cases of muscular dystrophy, 2 cases of polymyositis and all cases of juvenile dermatomyositis. No complement was observed in the control samples. Our results provide evidence for the sublytic formation of the membrane attack complex (MAC) on non-necrotic fibres in inflammatory myopathies and muscular dystrophy. This sublytic formation of the MAC may induce sublethal metabolic damage, mediated by calcium, and suggests a primary role of complement in muscle damage not only in inflammatory disorders but also muscular dystrophy.

Adolescent↗
PubMed1983

The normal occurrence of two molecular forms of the eight complement component (C8) and their concentrations in a family with C8 deficiency.

A haemolytically inactive alpha-mobile complement C8 component was identified by crossed immunoelectrophoresis in a 27-year-old male with recurrent meningococcal infections and absence of complement mediated serum haemolytic activity. The patient's serum/plasma contained no demonstrable normal C8. C8 inhibitor activity was not demonstrable in his serum and the alternative activation pathway appeared to be intact. Serum from the proband's parents and both siblings had normal total haemolytic complement activity and they were without symptoms. However, both the normal and the haemolytically inactive C8 protein were demonstrable in their sera. Their normal C8 concentrations were less than one-half of the normal C8 mean value and distinctly below the lowest value found in ten healthy persons. The concentration of the inactive C8 component in the proband's serum was roughly 1 . 5 times higher than that of the other family members, whereas its concentration in sera of the family members was between 1 . 8 and 2 . 5 times the highest concentration found in ten healthy donors, all of whom also had an alpha-mobile C8 in measurable quantities.

Adult↗
PubMed1985

Genetic polymorphism of complement component C8.

Extensive genetic polymorphism of complement component C8 was demonstrated by isoelectric focusing of serum or plasma samples followed by immunoblotting procedures. Using these methods, we could detect both alpha-gamma (C81) and beta (C82) chain polymorphisms in the same gel. Two-dimensional (2D) electrophoresis of C8 immunoprecipitates was used to obtain further information of the C8 patterns. Evidence was obtained that the C81 polymorphism resides in the structural gene of the C8 alpha chain. Both C8 systems show autosomal, chiefly codominant inheritance, and the distribution of phenotypes agrees with the Hardy-Weinberg equilibrium. Our findings suggest at least five different alleles in the C81 system; the gene frequencies of the two most common ones, C81*A and C81*B being 0.59 and 0.39, respectively. In C82 we found evidence for at least three codominant alleles, the gene frequencies for the two most common ones, C82*B and C82*A being 0.94 and 0.05, respectively. In addition, family studies disclosed the existence of a null allele, C82*Q0.

Alleles↗
PubMed2000

Human complement protein C8 gamma.

Human C8 gamma is a 22 kDa subunit of complement component C8, which is one of five components (C5b, C6, C7, C8, C9) that interact to form the cytolytic membrane attack complex (MAC) of complement. C8 contains three nonidentical subunits (alpha, beta, gamma) that are products of different genes. These subunits are arranged asymmetrically to form a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. C8 alpha and C8 beta are homologous to C6, C7 and C9 and together these proteins comprise what is referred to as the 'MAC protein family'. By comparison, C8 gamma is distinct in that it belongs to the lipocalin family of small, secreted proteins which have the common ability to bind small hydrophobic ligands. While specific roles have been identified for C8 alpha and C8 beta in the formation and function of the MAC, a function for C8 gamma and the identity of its ligand are unknown. This review summarizes the current status of C8 gamma structure and function and the progress made from efforts to determine its role in the complement system.

Amino Acid Sequence↗
PubMed1986

Antigenic crossreactivity of the alpha subunit of complement component C8 with the cysteine-rich domain shared by complement component C9 and low density lipoprotein receptor.

Complement component C9 contains two distinct cysteine-rich domains exhibiting high sequence resemblance to a domain present in the low density lipoprotein (LDL) receptor and epidermal growth factor precursor, respectively. Antibodies were raised against a peptide corresponding to the most conserved region of the LDL receptor/C9-homology segment. The antibodies were shown by immunoblotting to bind specifically to C9 but also to crossreact with C8 alpha, the alpha subunit of complement component C8. Moreover, a monoclonal antibody to a neoantigen present in polymerized C9 bound to an epitope exposed on C8 within the C5b-8 complex but buried in monomeric C8, suggesting that C8 and C9 undergo similar conformational changes during membrane-attack-complex assembly. Isolated C8 alpha-gamma exhibited the propensity to polymerize in the presence of Zn2+ and urea, as already demonstrated for C9. These data indicate that C8 alpha is closely related, both structurally and functionally, to C9.

Complement C8↗