Functional studies of the MACPF domain of human complement protein C8alpha reveal sites for simultaneous binding of C8beta, C8gamma, and C9.

Human C8 is one of five components of the membrane attack complex of complement (MAC). It contains three subunits (C8alpha, C8beta, C8gamma) arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. C8alpha, C8beta, and complement components C6, C7, and C9 form the MAC family of proteins. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. During MAC formation, C8alpha binds and mediates the self-polymerization of C9 to form a pore-like structure on target cells. The C9 binding site was previously shown to reside within a 52-kDa segment composed of the C8alpha N-terminal modules and MACPF domain (alphaMACPF). In the present study, we examined the role of the MACPF domain in binding C9. Recombinant alphaMACPF and a disulfide-linked alphaMACPF-gamma dimer were successfully produced in Escherichia coli and purified. alphaMACPF was shown to simultaneously bind C8beta, C8gamma, and C9 and form a noncovalent alphaMACPF.C8beta.C8gamma.C9 complex. Similar results were obtained for the recombinant alphaMACPF-gamma dimer. This dimer bound C8beta and C9 to form a hemolytically active (alphaMACPF-gamma).C8beta.C9 complex. These results indicate that the principal binding site for C9 lies within the MACPF domain of C8alpha. They also suggest this site and the binding sites for C8beta and C8gamma are distinct. alphaMACPF is the first human MACPF domain to be produced recombinantly and in a functional form. Such a result suggests that this segment of C8alpha and corresponding segments of the other MAC family members are independently folded domains.

Crystal structure of human complement protein C8gamma at 1.2 A resolution reveals a lipocalin fold and a distinct ligand binding site.

C8gamma is a 22-kDa subunit of human C8, which is one of five components of the cytolytic membrane attack complex of complement (MAC). C8gamma is disulfide-linked to a C8alpha subunit that is noncovalently associated with a C8beta chain. In the present study, the three-dimensional structure of recombinant C8gamma was determined by X-ray diffraction to 1.2 A resolution. The structure displays a typical lipocalin fold forming a calyx with a distinct binding pocket that is indicative of a ligand-binding function for C8gamma. When compared to other lipocalins, the overall structure is most similar to neutrophil gelatinase associated lipocalin (NGAL), a protein released from granules of activated neutrophils. Notable differences include a much deeper binding pocket in C8gamma as well as variation in the identity and position of residues lining the pocket. In C8gamma, these residues allow ligand access to a large hydrophobic cavity at the base of the calyx, whereas corresponding residues in NGAL restrict access. This suggests the natural ligands for C8gamma and NGAL are significantly different in size. Cys40 in C8gamma, which forms the disulfide bond to C8alpha, is located in a partially disordered loop (loop 1, residues 38-52) near the opening of the calyx. Access to the calyx may be regulated by movement of this loop in response to conformational changes in C8alpha during MAC formation.

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.

Isolation and characterization of the eighth component of the bovine complement system.

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.

Purification and characterization of the eighth and ninth components of carp complement.

Complement components corresponding to mammalian C8 and C9 were isolated from carp (Cyprinus carpio) serum. Carp C8 (M(r) 146,000) proved to be a gamma-globulin composed of three polypeptide chains (alpha-chain, M(r) 62,000; beta-chain, M(r) 62,000; gamma-chain, M(r) 22,000). The alpha-chain was disulfide-linked to the gamma-chain and the beta-chain was non-covalently associated with the alpha-gamma chain, in fair agreement with mammalian C8. However, the N-terminal amino acid sequences of the three subunits showed no homology with those of human C8. Carp C9 was an alpha-globulin composed of a single polypeptide (M(r) 91,000) and the N-terminus was blocked. Carp serum depleted of C8 did not hemolyse either carp antibody-sensitized sheep erythrocytes or non-sensitized rabbit erythrocytes, while C9-depleted carp serum did not hemolyse the former, but did hemolyse the latter target cells, as in the case of C9-depleted human serum.

Interaction between apolipoproteins A-I and A-II and the membrane attack complex of complement. Affinity of the apoproteins for polymeric C9.

We have previously observed enhanced binding of HDL and apolipoproteins A-I and A-II to human endothelial cells exposed to activated complement. Induction of these binding sites required complement activation through C9, suggesting a specific role for the C9 component of the C5b-9 complex. We now report that specific and saturable binding sites for apoA-I and -A-II are expressed by C9 polymers (polyC9), whereas little binding was observed to native monomeric C9. These data suggested an interaction of the apoproteins with a site(s) which is exposed only upon C9 polymerization, and also suggested that binding of the apoproteins to this new site might interfere with assembly of C9 into the polyC9 tubule and insertion into the cell membrane. ApoA-I was found to inhibit zinc-catalyzed polymerization of C9 in a concentration-dependent fashion. Formation of SDS-resistant C9 polymers was completely inhibited at apoA-I or -A-II concentrations > or = 5 microM. ApoA-I also produced a concentration-dependent inhibition of C9 incorporation into C5b-9 complexes on endothelial cells, which was accompanied by a corresponding decrease in SDS-resistant C9 polymers associated with the cell membrane. In summary, the ability of the HDL apoproteins A-I and A-II to interact with an activation-dependent conformer(s) of the C9 component of the C5b-9 complex appears to explain the expression of HDL binding sites on endothelial cells exposed to complement. These apoproteins are also inhibitors of C9 polymerization, which may underlie the protective effect of HDL for blood cells exposed to activated complement.

Purification of C8 and C9 from rat serum.

A procedure based on modifications of published methods for human proteins for the isolation of rat C8 and C9 from one batch of serum is described. The procedure allows the rapid, large-scale isolation of pure and haemolytically active proteins. Rat C9 had a slightly higher molecular weight than human C9 on SDS-PAGE and similar isoelectric point. Rat C8 differed from human C8 in the molecular weight of the gamma chain (23,000 and 21,000 kD respectively), and on isoelectric focusing (pI rat C8: 6.5-6.9; pI human C8: 7.4-7.9).

Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids.

We have previously reported the isolation of a membrane-attack-complex-inhibiting protein (MIP) from human erythrocyte membranes [Watts, Patel & Morgan (1987) Complement 4, 236] and the production of polyclonal antibodies to this protein. Here we report the identification in plasma, urine, saliva and cerebrospinal fluid of a protein immunochemically identical with the membrane-derived MIP. The protein has been isolated from plasma by immunoaffinity chromatography on an anti-(erythrocyte MIP)-Sepharose column and shown by SDS/polyacrylamide-gel electrophoresis to be of similar molecular mass to the erythrocyte protein (55 kDa non-reduced and 65 kDa under reducing conditions). Monoclonal antibodies have been raised against plasma MIP and used to establish a two-site enzyme-linked immunoadsorbent assay, enabling quantification of MIP in plasma, urine and cerebrospinal fluid. Plasma MIP, though not able to incorporate spontaneously into membranes, was deposited on heterologous and homologous erythrocyte membranes during complement activation in a C8-dependent manner. Depletion of MIP from plasma resulted in enhancement of the lytic capacity of the plasma on heterologous erythrocytes.

C8 reference typing report and nomenclature recommendation.

Using two different typing techniques (i.e. polyacrylamide gel isoelectric focusing (PAGIF) with Western blot and SDS-polyacrylamide gel electrophoresis of precipitated C8 under nonreducing conditions with Western blot), the following observations were made during the reference typing for C81 (C8A). The Japanese variant A1J is probably identical with A1Cauc, whereas B1J is definitely different from B1Cauc and could therefore provisionally be named HB3'. Variant 'A2' from Japan is focused in an intermediate position, but different from M1 and could be named 'M2'. Both variants possess normal A subunits. B2 from Japan is clearly different from B1Cauc and should retain its designation. In PAGIF, its subunit has a position more anodal than A. Summarizing these results, more information is needed before a final nomenclature can be proposed.

Differential functional expression of the C8 subunits. Primary role of C8 beta in assembly of intact C8.

The eighth component of C (C8) is composed of two subunits C8 beta and C8 alpha-gamma, which are non-covalently bound in a 1/1 ratio in the intact molecule. The genes encoding the polypeptide chains composing the subunits demonstrate close genetic linkage. To assess the functional expression of these genes at the protein level, normal human serum and C8-deficient sera were electrophoresed in native polyacrylamide gels following which C8, C8 beta, and C8 alpha-gamma were detected using hemolytic overlays. These experiments demonstrated that normal sera contained free C8 alpha-gamma in addition to intact C8. Free C8 alpha-gamma was not observed when C8 was reconstituted by mixing C8 beta-deficient serum with C8 alpha-gamma-deficient serum in a ratio optimized for C8 activity, suggesting that the free C8 alpha-gamma observed in normal serum was not due to dissociation of intact C8. Inasmuch as this technique did not adequately separate C8 and C8 beta, sera were also examined by anion exchange chromatography. C8 alpha-gamma-deficient serum contained C8 beta in a single peak in the 1.4 ms/cm fall through. C8 beta-deficient serum contained a major peak of C8 alpha-gamma at 7.1 ms/cm and a lesser peak coeluting with C9 at 9.5 ms/cm. Normal serum contained both intact C8 eluting between 2.4 to 5.5 ms/cm and C8 alpha-gamma eluting at 7.1 ms/cm. Free C8 beta was not detectable in normal serum indicating that free C8 alpha-gamma was not due to C8 dissociation. Mixing aliquots from the chromatographic peak of C8 beta activity with the peaks of C8 alpha-gamma activity in C8 beta-deficient serum or in normal serum generated intact C8 hemolytic activity. Non-reducing SDS-PAGE and Western blotting with anti-C8 confirmed the presence of antigenic material of appropriate m.w. in each peak. These findings demonstrate that serum contains excess C8 alpha-gamma relative to C8 beta, despite the equimolar presence of the subunits in intact C8. Thus the availability of C8 beta determines the quantity of C8 produced. Further, these data suggest the possibility that the C8 structural genes may be differentially expressed despite their close genetic linkage.

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit.

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.

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.

The eighth component of human complement: evidence that it is an oligomeric serum protein assembled from products of three different genes.

The eighth component of human complement (C8) consists of three nonidentical subunits arranged asymmetrically as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. Genetic studies of C8 polymorphisms established that alpha-gamma and beta are encoded at different loci. Implicit in this finding was the existence of two different genes and the likelihood that alpha-gamma would be synthesized in single-chain precursor form. However, recent characterization of cDNA clones revealed separate mRNAs for human alpha and beta but no evidence of a single-chain precursor for alpha-gamma. A cDNA clone containing the entire coding region for human gamma has now been characterized, and its sequence supports the existence of a separate gamma mRNA. Included are a consensus translation initiation sequence, an apparent initiation methionine, and a signal peptide. By use of cDNA probes specific for human alpha, beta, or gamma, analysis of poly(A) RNA from normal baboon liver revealed separate mRNAs of 2.5, 2.6, and 1.0 kilobases (kb), respectively. Parallel analysis of poly(A) RNA from rat liver identified mRNAs of 3.4, 2.3, and 0.9 kb. These results argue against the possibility that C8 is assembled from products of two different genes (alpha-gamma and beta) and suggest it is comprised of three different gene products (alpha, beta, and gamma) that undergo both covalent and noncovalent association to yield the mature protein.

Restriction in the lytic efficiency of complement of different erythrocyte targets: a re-examination of the activities of horse C8 and C9.

Differences in the lytic efficiency of different complement sources have frequently been observed. This effect has been shown to be related to both the species of the target erythrocyte and the species composition of terminal complement components within the 5b-9 membrane attack complex. The majority of studies have indicated that the source of C9 is critical in controlling the range of erythrocyte species that can be lysed efficiently. One exception to this general finding was the report by Lachmann et al., 1973 (Immunology 24, 135-145), using horse serum as a complement source. In that study, horse C8 rather than C9 was implicated as the critical component. In this study, we have re-examined this observation and find that the restricted hemolytic potential of horse complement correlates absolutely with the presence of horse C9. The reason for the differences between our findings and those of the earlier study are discussed.

Analysis of the specific association of the eighth and ninth components of human complement: identification of a direct role for the alpha subunit of C8.

The basis for the physical association between C8 and C9 in solution was examined by isolating the noncovalently associated alpha-gamma and beta subunits of C8 and determining their respective affinities for C9. Results indicate that only alpha-gamma associates with C9 and this association, though reversible, is complete at near equimolar ratios of each component. Further experiments using purified alpha or gamma revealed that only alpha was capable of forming a stable complex with C9. Although the strength of this interaction was dependent on salt concentration, association was observed in buffer of physiological ionic strength and in human serum. These results establish that the domain on C8 responsible for interaction with C9 is located entirely within alpha. In related experiments, addition of beta to performed dimers of either (alpha-gamma + C9) or (alpha + C9) resulted in complete association of this subunit. These particular results indicate that there are two physically distinct sites on alpha that separately mediate association of alpha with beta and with C9. Furthermore, occupation of one site does not impair interaction at the other.

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.

Genetic deficiency of the alpha-gamma-subunit of the eighth complement component in the rabbit.

Genetic deficiency of the alpha-gamma-subunit of the eighth complement component (C8 alpha-gamma) was found in a strain of the New Zealand White rabbit. The serum of this deficient rabbit lacked the immunochemical and functional alpha-gamma-subunit of C8. Mating tests indicate that the C8 alpha-gamma deficiency is transmitted as a simple autosomal recessive trait with the following physiologic characteristics. The body weight at the first week of life, mature weight, and litter size of the deficient animals were smaller than those of heterozygous and normal ones. In addition, survival rates for the first 3 mo of life of the deficient animals tended to be lower than those of heterozygous and normal littermates.

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)

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.