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PubMed1987

Complementary DNA and derived amino acid sequence of the beta subunit of human complement protein C8: identification of a close structural and ancestral relationship to the alpha subunit and C9.

A cDNA clone encoding the beta subunit (Mr 64,000) of the eighth component of complement (C8) has been isolated from a human liver cDNA library. This clone has a cDNA insert of 1.95 kilobases (kb) and contains the entire beta sequence [1608 base pairs (bp)]. Analysis of total cellular RNA isolated from the hepatoma cell line HepG2 revealed the mRNA for beta to be approximately 2.5 kb. This is similar to the message size for the alpha subunit of C8 and confirms the existence of different mRNAs for alpha and beta. This finding supports genetic evidence that alpha and beta are encoded at different loci. Analysis of the derived amino acid sequence revealed several membrane surface seeking segments that may facilitate beta interaction with target membranes during complement-mediated cytolysis. Determination of the carbohydrate composition indicated 1 or 2 asparagine-linked but no O-linked oligosaccharide chains. Comparison of the beta sequence to that reported for alpha in the preceding paper [Rao, A. G., Howard, O. M. Z., Ng, S. C., Whitehead, A. S., Colten, H. R. & Sodetz, J. M. (1987) Biochemistry (preceding paper in this issue)] and to that of human C9 revealed a striking homology between all three proteins. For beta and alpha, the overall homology is 33% on the basis of identity and 53% when conserved substitutions are allowed. For beta and C9, the values are 26% and 47%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Inhibition of the formation of the complement membrane-attack complex by a monoclonal antibody to the complement component C8 alpha subunit.

The effect of nine monoclonal antibodies to complement component C8 on the interaction of C9 with preformed cell-surface C5b-8 complexes and on the functional insertion of C8 into the membrane-attack complex (MAC) was investigated. None of the antibodies prevented C9 insertion into a preformed C5b-8 complex. One antibody (F1) directed to the C8 alpha subunit clearly inhibited formation of a functional MAC. It is proposed that this antibody prevents the C8 alpha subunit unfolding and distorting the bilayer to allow C9 insertion.

Animals↗
PubMed1989

Chromosomal assignment of genes encoding the alpha, beta, and gamma subunits of human complement protein C8: identification of a close physical linkage between the alpha and the beta loci.

The eighth component of human complement (C8) is a serum protein containing three nonidentical subunits (alpha, beta, gamma) that are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. In earlier genetic studies, electrophoretic analysis of C8 protein polymorphisms revealed several allelic variants of alpha-gamma and beta. These were governed by separate loci designated C8A and C8B for alpha-gamma and beta, respectively. Genetic linkage analyses indicated that these loci were linked to each other and to chromosome 1 marker loci PGM1 and Rh, but it was unclear at the time if C8A was a single locus coding for a single-chain precursor form of alpha-gamma or if separate loci existed for alpha and gamma. Since evidence now indicates that alpha, beta, and gamma are encoded by separate genes, cDNA probes corresponding to each subunit were used to make direct assignments of the individual loci. Analysis of somatic cell hybrids revealed that only the alpha and beta loci are located on chromosome 1. Parallel analysis of genomic DNA digests using 5' and 3'-specific cDNA probes showed they are physically linked (less than 2.5 kb) and oriented 5' alpha-beta 3'. Further probing of the hybrid panel revealed that gamma is located on chromosome 9q. Thus, the observed genetic linkage of alpha-gamma to beta must be determined solely by alpha. In accordance with these findings, the C8 loci should now be designated C8A, C8B, and C8G for alpha, beta and gamma, respectively.

Animals↗
PubMed1994

Genomic structure of the human complement protein C8 gamma: homology to the lipocalin gene family.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target cells. It contains three subunits (C8 alpha, C8 beta, C8 gamma) which are encoded in separate genes. In relation to other proteins of the complement system, C8 gamma is unusual in that it is not structurally related to any other component nor does it have an obvious function. Based on weak but significant sequence similarity, it is proposed to be a member of the lipocalin family of widely distributed proteins that bind and transport small hydrophobic ligands. In this study, the human C8 gamma gene has been characterized and found to contain seven exons spanning approximately 1.8 kb. S1 nuclease and anchored PCR were used to identify the transcription initiation site. This site is preceded by putative regulatory elements that include two SP1 binding sites, several glucocorticoid response elements, and two SV40 enhancer core consensus sequences. A comparison to genes of other lipocalins reveals a remarkably close correlation in exon number, lengths, and phases. A close correspondence in exon boundaries is also observed and suggests that C8 gamma contains the same discrete structural elements that define the characteristic beta-barrel shape of the lipocalins. These results establish that C8 gamma is indeed ancestrally related to the lipocalin family and strengthens the likelihood that its role in the complement system is to bind an as yet unidentified ligand.

Amino Acid Sequence↗
PubMed1987

Complementary DNA and derived amino acid sequence of the alpha subunit of human complement protein C8: evidence for the existence of a separate alpha subunit messenger RNA.

The entire amino acid sequence of the alpha subunit (Mr 64,000) of the eighth component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire alpha coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A) sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of approximately 2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for alpha and argues against the occurrence of a single-chain precursor form of the disulfide-linked alpha-gamma subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. These occur in a cysteine-free region of the subunit and may constitute the structural basis for alpha interaction with target membranes. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

The preparation and characterization of monoclonal antibodies to human complement component C8 and their use in purification of C8 and C8 subunits.

1. Ten mouse monoclonal antibodies to human complement component C8 were prepared. It was found that six of these antibodies reacted with the alpha-subunit, two with the beta-subunit and two with the gamma-subunit, when assessed by immunoblotting after separation of C8 subunits by SDS/polyacrylamide-gel electrophoresis. 2. Epitope analysis of the ten monoclonal antibodies in a competitive binding assay showed that the six antibodies to the alpha-subunit could be classified in four overlapping epitope groups. The antibodies to the beta- and gamma-subunits bound to a single antigenic site on each, but also cross-reacted with the antigenic sites on the alpha-subunit. 3. Monoclonal anti-C8 immunoaffinity columns were used to purify C8 from fresh human plasma and to prepare C8-depleted serum. Immunoaffinity purified C8 was biologically active when assessed by using haemolysis assays of sheep and rabbit erythrocytes. 4. Salt elution was used to purify either alpha gamma- or beta-subunits when C8 was respectively bound to an anti-beta or anti-alpha immunoaffinity column. The purified subunits reconstituted C8-depleted serum when added together in a haemolysis assay.

Antibodies, Monoclonal↗
PubMed2006

Cloning and phylogenetic analysis of the alpha subunit of the eighth complement component (C8) in rainbow trout.

The alpha subunit of the eighth complement component (C8) is a single-chain plasma glycoprotein which functions in the cytolytic process mediated by the complement system through a sequence of polymerization reactions with other terminal components. We have previously isolated and characterized the C8beta and C8gamma subunits of the eighth complement component in rainbow trout (Oncorhynchus mykiss). Here, we report the primary sequence, the tissue expression profile, the domain architecture and the phylogenetic analysis of the trout C8alpha gene. The deduced amino acid sequence of the trout C8alpha gene exhibits 44 and 43% identity with human and frog orthologs, respectively. The domain architecture of the trout C8alpha resembles that of mammalian orthologs, and the cysteine backbone shows a high degree of conservation. The trout C8alpha shows a similar expression profile with that of trout C8beta and C8gamma, pointing to the liver as the main source of the C8 genes expression. Although the presence of a fully developed lytic pathway of complement system is expected in teleost, this is the first report of the C8alpha gene in an organism other than mammalian.

Amino Acid Sequence↗
PubMed1987

The homology of complement factor C8 gamma chain and alpha-1-microglobulin.

The sequence of the complement factor C8 gamma chain shares a remarkable degree of similarity with that of alpha-1-microglobulin, a member of the alpha-2u-globulin superfamily. This superfamily comprises a diverse group of distantly related animal proteins possessing characteristic structural features and similar functions. Comparison of the C8 gamma chain with these proteins supports its homology to them and suggests a possible functional role.

Alpha-Globulins↗
PubMed1995

Genomic organization of human complement protein C8 alpha and further examination of its linkage to C8 beta.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma) encoded by separate genes. C8 alpha and C8 beta are linked on chromosome 1p32, whereas C8 gamma is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 alpha gene. The gene contains at least 11 exons spanning approximately 70 kb of DNA. When compared to C6, C8 beta and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 alpha and C8 beta linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5 kb) and in a 5' alpha-beta 3' orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.

Amino Acid Sequence↗
PubMed1994

Human complement component C8. Molecular basis of the beta-chain polymorphism.

The beta-chain of human complement component C8 exhibits a structural genetic polymorphism: using isoelectric focusing two major allotypes can be identified (C8B B ('basic') and C8B A ('acidic')). In the present report we describe a sequence polymorphism of the C8B gene (codon 63: AGA-->GGA) and demonstrate that the resulting amino acid substitution (Arg-->Gly) consistently differentiates between the two common charge variants of the C8 beta chain; the C8B B allotype is characterized by an Arg and the C8B A allotype by a Gly residue in position 63 of the C8 beta polypeptide chain.

Amino Acid Sequence↗
PubMed1994

Characterization of rabbit complement component C8. Functional evidence for the species-selective recognition of C8 alpha by homologous restriction factor (CD59).

Protection of host cells from lysis by the C5b-9 cytolytic complex is provided by a membrane-associated protein (CD59) that interacts with homologous C8 and C9 during C5b-9 assembly. This interaction restricts normal formation and function of the complex, thereby protecting cells from attack by homologous C. In this study, rabbit C8 was purified and characterized and used to investigate the role of the individual C8 subunits in homologous restriction and the basis for species selectivity by human CD59. Exchanging the disulfide-linked C8 alpha-gamma dimer in human C8 with one from rabbit was found to be sufficient to overcome restriction by human Es. Activity of the hybrid C8 and rabbit C8 toward these target cells was equivalent, thus establishing that restriction is not dependent on the species of C8 beta. Because C8 gamma was previously shown to have no role in restriction, these results suggest that within C8, structural determinant(s) recognized by CD59 reside solely on C8 alpha. Sequences determined from full-length cDNA clones for rabbit C8 alpha, C8 beta, and C8 gamma support this conclusion. All three subunits are strikingly similar to human with regard to length, m.w., N- and C-terminal residues, conserved cysteines, and overall sequence. However, when sequences are compared under high stringency, a single segment of extended sequence dissimilarity is detected between the two species of C8 alpha. Within human C8 alpha, this 37-residue segment resides near the putative membrane-interacting region and may constitute a human CD59 recognition site.

Amino Acid Sequence↗
PubMed1990

The homologous restriction factor is immunologically related to complement components C8 and C9 and to lymphocyte pore-forming protein perforin through cysteine-rich domains.

The 65 kDa C8-binding protein or homologous restriction factor (C8bp/HRF) protects cells from complement (C)-mediated lysis by binding to C8 and abrogating lytic channel formation. Human C8bp/HRF is shown here to be immunologically related to human C8 and C9 and to murine lymphocyte poreforming protein (PFP, perforin). Polyclonal antibodies raised against purified C8, C9 and perforin react with C8bp/HRF. The antigenic epitopes shared by these four proteins are limited to cysteine-rich or disultide bridge-masked domains. Only complement proteins or perforin that have been disulfide-reduced elicit the production of cross-reactive antibodies when used as immunogens. Analogously, only C8bp/HRF that has been disulfide-reduced reacts with these antibodies. These results suggest that C8bp/HRF may belong to the complement/perforin supergene family. The function of homologous domains shared by these four proteins remains to be elucidated.

Animals↗
PubMed1999

Chimeric and truncated forms of human complement protein C8 alpha reveal binding sites for C8 beta and C8 gamma within the membrane attack complex/perforin region.

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.

Animals↗
PubMed2005

The gamma subunit of the eighth complement component (C8) in rainbow trout.

Of the 35 proteins, enzymes, receptors and regulatory components of the complement system, C8gamma is unique in that it is the only lipocalin. C8gamma is a subunit of the C8 molecule, which is one of the five components (C5b, C6, C7, C8 and C9) that interact as a consequence of complement activation to form the membrane attack complex. Until now, C8gamma has been characterized only in mammalian species. In order to elucidate the phylogeny of this molecule, we have cloned the C8gamma subunit in rainbow trout (Oncorhynchus mykiss), a teleost fish representing a critical point in the evolutionary divergence of the complement system. The deduced amino acid sequence of trout C8gamma shows significant identity (37%) to the human C8gamma homolog and much lower to the other known lipocalins. The lipocalin domain is present and all the cysteine residues are conserved. The trout C8gamma gene is probably present as a single copy in the trout genome showing a differential expression pattern among tissues investigated.

Amino Acid Sequence↗
PubMed1989

Biotinylation: a simple method for labelling complement component C8 with preservation of functional activity.

Biotinylation of human C8 with the water-soluble biotin derivative biotinylamidohexanoic acid, N-hydroxysulfosuccinimide ester is an excellent method for labelling this terminal complement component with preservation of its functional activity. The biotinylated product can be detected both in native form and also following its incorporation into the terminal complement complexes. Detection assays include Western blotting, crossed immunoblotting, ELISA, and immunocytochemistry. Biotinylation is an attractive alternative method for labelling C8 and may be used for detecting and quantifying C8 and C5b-9 complexes in their soluble and membrane-bound forms.

Biotin↗
PubMed1993

Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9.

Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.

Clusterin↗