Structural basis of soluble membrane attack complex packaging for clearance.

Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane ß-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.

Structural compliance: A new metric for protein flexibility.

Proteins are the active players in performing essential molecular activities throughout biology, and their dynamics has been broadly demonstrated to relate to their mechanisms. The intrinsic fluctuations have often been used to represent their dynamics and then compared to the experimental B-factors. However, proteins do not move in a vacuum and their motions are modulated by solvent that can impose forces on the structure. In this paper, we introduce a new structural concept, which has been called the structural compliance, for the evaluation of the global and local deformability of the protein structure in response to intramolecular and solvent forces. Based on the application of pairwise pulling forces to a protein elastic network, this structural quantity has been computed and sometimes is even found to yield an improved correlation with the experimental B-factors, meaning that it may serve as a better metric for protein flexibility. The inverse of structural compliance, namely the structural stiffness, has also been defined, which shows a clear anticorrelation with the experimental data. Although the present applications are made to proteins, this approach can also be applied to other biomolecular structures such as RNA. This present study considers only elastic network models, but the approach could be applied further to conventional atomic molecular dynamics. Compliance is found to have a slightly better agreement with the experimental B-factors, perhaps reflecting its bias toward the effects of local perturbations, in contrast to mean square fluctuations. The code for calculating protein compliance and stiffness is freely accessible at https://jerniganlab.github.io/Software/PACKMAN/Tutorials/compliance.

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers.

The membrane attack complex (MAC) is one of the immune system's first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant ß-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ß-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

Comprehensive Proteoform Characterization of Plasma Complement Component C8αßγ by Hybrid Mass Spectrometry Approaches.

The human complement hetero-trimeric C8αßγ (C8) protein assembly (~ 150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~ 20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions. Their native MS analysis revealed even more detailed structural micro-heterogeneity on C8. Subsequent peptide-centric analysis, by proteolytic digestion of C8 and LC-MS/MS, provided site-specific quantitative profiles of different types of C8 glycosylation. Combining all this data provides a detailed specification of co-occurring C8 proteoforms, including experimental evidence on N-glycosylation, C-mannosylation, and O-glycosylation. In addition to the known N-glycosylation sites, two more N-glycosylation sites were detected on C8. Additionally, we elucidated the stoichiometry of all C-mannosylation sites in all the thrombospondin-like (TSP) domains of C8α and C8ß. Lastly, our data contain the first experimental evidence of O-linked glycans located on C8γ. Albeit low abundant, these O-glycans are the first PTMs ever detected on this subunit. By placing the observed PTMs in structural models of free C8 and C8 embedded in the MAC, it may be speculated that some of the newly identified modifications may play a role in the MAC formation. Graphical Abstract ᅟ.

Identification and expression of complement component C8α, C8ß and C8γ gene in half-smooth tongue sole (Cynoglossus semilaevis) and C8α recombinant protein antibacterial activity analysis.

Complement component C8, which mediates membrane attack complex formation and bacterial lysis, plays important roles in the complement system. The cDNA sequences of the C8α, C8ß and C8γ genes were cloned from half-smooth tongue sole (Cynoglossus semilaevis). Full-length cDNA of CsC8α (C8α of C. semilaevis), CsC8ß and CsC8γ was 1990, 2219 and 886 bp, respectively, which contained open reading frames of 1797, 1749 and 666 bp, encoding 598, 582 and 221 amino acids, respectively. The deduced proteins of CsC8α, CsC8ß and CsC8γ showed the closest amino acid similarity to C8α (73%) of Siniperca chuatsi, C8ß (76%) of Oryzias latipes and C8γ (72%) of Takifugu rubripes, respectively. The highest expression level of CsC8α, CsC8ß and CsC8γ among the 13 normal tissues was observed in liver tissue, followed by much lower levels in other tissues. After infection with Vibrio anguillarum, CsC8α, CsC8ß and CsC8γ were significantly up-regulated in all of the detected tissues, including the intestine, liver, gill, head kidney, blood and spleen. Then, a recombinant expression plasmid was constructed, and the recombinant CsC8α protein was expressed in GS115 pichia pastoris yeast. Furthermore, to investigate the biological functions of recombinant CsC8α, an antibacterial assay was performed, and the results showed that recombinant CsC8α obviously inhibited growth of V. anguillarum, Edwardsiella tarda and Vibrio parahaemolyticus. Taken together, these results suggest that CsC8α, CsC8ß and CsC8γ may play important roles in the immune defense of C. semilaevis.

Effect of ammonia-N and pathogen challenge on complement component 8α and 8ß expression in the darkbarbel catfish Pelteobagrus vachellii.

The complement components C8α and C8ß mediate the formation of the membrane attack complex (MAC) to resist pathogenic bacteria and play important roles in innate immunity. Full-length complement C8α (Pv-C8α) and C8ß (Pv-C8ß) cDNA were identified in the darkbarbel catfish Pelteobagrus vachellii, and their mRNA expression levels were analyzed after ammonia-N and pathogen treatment. The Pv-C8α gene contained 1983 bp, including a 1794-bp open reading frame (ORF) encoding 598 amino acids. The Pv-C8ß gene contained 1952 bp, including a 1761-bp ORF encoding 587 amino acids. Pv-C8α and Pv-C8ß had the highest amino acid identity with rainbow trout Oncorhynchus mykiss C8α (62%) and Japanese flounder Paralichthys olivaceus C8ß (83%), respectively. Sequence analysis indicated that both Pv-C8α and Pv-C8ß contained a thrombospondin type-1 (TSP1) domain, a low-density lipoprotein receptor class A (LDLR-A) domain, a membrane attack complex/perforin (MACPF) domain and an epidermal growth factor-like (EGF-like) domain. In addition, Pv-C8α and Pv-C8ß were mainly distributed in the liver, head kidney, spleen, and eggs. Under ammonia-N stress, the Pv-C8α and Pv-C8ß mRNA levels significantly decreased (P < 0.05), with minimum levels, respectively, attained at 24 and 48 h in the liver, 48 and 24 h in the head kidney, and 24 and 24 h in the spleen. After Aeromonas hydrophila challenge, the Pv-C8α and Pv-C8ß mRNA levels significantly increased (P < 0.05), with maximum levels, respectively, attained at 48 and 24 h in the liver, 24 and 48 h in the head kidney, and 48 and 48 h in the spleen. The present study indicated that Pv-C8α and Pv-C8ß exhibited important immune responses to infection and that ammonia-N in water decreased the immune responses of Pv-C8α and Pv-C8ß.

Fabrication of polyion complex vesicles with enhanced salt and temperature resistance and their potential applications as enzymatic nanoreactors.

Integrating catalytic functions into polymeric vesicles through enzyme entrapment is appealing for bioreactor fabrication, yet there are critical issues regarding the regulation of solute transport through membranes and enzyme loading without denaturation. Polyion complex vesicles (PICsomes) with semipermeable membranes and the propensity to form in water can overcome these issues; however, cross-linking is required for sufficient physiological stability. Herein, we report the first successful fabrication of non-cross-linked PICsomes with sufficient stability at physiological salinity and temperature by tuning the hydrophobicity of the aliphatic side chains in the pendant group of the constituent polyelectrolytes. Dynamic light scattering and transmission electron microscopy revealed that the intervesicular fusion and disintegration of the PICsomes was prevented and a narrow distribution was maintained at physiological salinity and temperatures. Furthermore, their application as enzymatic nanoreactors was verified even in the presence of proteases. As such, the potential utility of the PICsomes in biomedical fields was established.

Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum).

Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.

Mapping the intermedilysin-human CD59 receptor interface reveals a deep correspondence with the binding site on CD59 for complement binding proteins C8alpha and C9.

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.

Structure of human C8 protein provides mechanistic insight into membrane pore formation by complement.

C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8α, C8ß, and C8γ. The C6, C7, C8α, C8ß, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N- and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8α and C8ß are located on the periphery of C8 and not likely to interact with the target membrane. The C8γ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8α and C8ß are related by a rotation of ∼22° with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane ß-hairpins to form a circular pore.

Structure of human complement C8, a precursor to membrane attack.

Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8ß-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus.

A disulfide linked model of the complement protein C8gamma complexed with C8alpha indel peptide.

In a recent study C8gamma (complement protein) with Cys40Ala substitution and a C8alpha derived peptide with Cys164Ala substitution were co-crystallized and their binding mode was revealed. Computer modeling and molecular dynamics simulations were performed in order to check the hypothesis that the residues Ala164 of C8alpha and Ala40 of C8gamma occupied the right position if cysteine residues were in their place for disulfide bonding. Substitution of these two alanine residues with cysteine along with disulfide bond creation via molecular modeling and subsequent molecular dynamics simulation of the complex corroborated the hypothesis, which was also confirmed from recent crystallographic data. Average RMSD between backbone atoms of the indel peptide during the MD trajectory in comparison with the corresponding sequence of crystal structure of the C8alpha/gamma complex was found only 0.085 nm.

Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit.

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that assemble on bacterial membranes to form a porelike structure referred to as the "membrane attack complex" (MAC). C8 contains three genetically distinct subunits (C8 alpha, C8 beta, C8 gamma) arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. C6, C7 C8 alpha, C8 beta, and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8 gamma subunit is unrelated and belongs to the lipocalin family of proteins that display a beta-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8 alpha MACPF domain were recently reported and both display a fold similar to those of the bacterial pore-forming cholesterol-dependent cytolysins (CDCs). In the present study, we determined the crystal structure of the human C8 alpha MACPF domain disulfide-linked to C8 gamma (alphaMACPF-gamma) at 2.15 A resolution. The alphaMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8 gamma. One is in a previously characterized 19-residue insertion (indel) in C8 alpha and fills the entrance to the putative C8 gamma ligand-binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8 gamma beta-barrel. The latter interaction induces conformational changes in alphaMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X(6)-G-G in alphaMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.

Crystal structure of complement protein C8gamma in complex with a peptide containing the C8gamma binding site on C8alpha: implications for C8gamma ligand binding.

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex. It contains three genetically distinct subunits; C8alpha and C8gamma form a disulfide-linked C8alpha-gamma heterodimer that is noncovalently associated with C8beta. The C8alpha subunit is homologous to C8beta, C6, C7 and C9 and together they form the MAC family of proteins. By contrast, C8gamma is the only lipocalin in the complement system. Like other lipocalins, it has a core beta-barrel structure forming a calyx with a distinct binding pocket for a small and as yet unidentified ligand. The binding site on C8alpha for C8gamma was previously localized to a 19-residue segment which contains an insertion (indel) that is unique to C8alpha. Included in the indel is C8alpha Cys 164 which links to Cys 40 in C8gamma. In the present study, C8gamma containing a C40A substitution was co-crystallized with a synthetic indel peptide containing the equivalent of a C8alpha C164A substitution. The X-ray crystal structure shows that the indel peptide completely fills the upper portion of the putative C8gamma ligand binding pocket and is in contact with all four loops at the calyx entrance. The lower part of the C8gamma cavity is either unoccupied or contains disordered solvent. The validity of the structure is supported by the spatial arrangement of C8alpha Ala 164 in the peptide and C8gamma Ala 40, which are within disulfide-bonding distance of each other. Corresponding studies in solution indicate the C8gamma ligand binding site is also occupied by the indel segment of C8alpha in whole C8. These results suggest a role for C8alpha in regulating access to the putative C8gamma ligand binding site.

Structure of C8alpha-MACPF reveals mechanism of membrane attack in complement immune defense.

Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8alpha at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane beta hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8alpha is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion.

Crystal structure of CD59: implications for molecular recognition of the complement proteins C8 and C9 in the membrane-attack complex.

Human CD59 is a small membrane-bound glycoprotein that functions as an inhibitor of the membrane-attack complex (MAC) of the complement system by binding the complement proteins C8 and C9. The crystal structure of a soluble construct of CD59 has been determined to 2.1 A resolution. When compared with previous models of CD59 determined using NMR, some interesting differences are noted, including the position of helix alpha1, which contributes to the binding surface for C8 and C9. Interestingly, the crystal structure superimposes more closely with an updated NMR model of CD59 that was produced using Monte Carlo minimization, including helix alpha1. Mapping of mutations associated with enhanced or lowered inhibitory function of CD59 show the binding region to be located in a crevice between alpha1 and a three-stranded beta-sheet, as has been identified previously. Residues in the core of this region are well ordered in the electron density, in part owing to a network of stabilizing covalent and noncovalent interactions, and manifest an interesting 'striped' distribution of hydrophobic and basic residues. Docking of the same peptide that was modeled previously into the NMR structure shows that Arg55, which has been postulated to exist in 'open' and 'closed' positions, is intermediate in position between these two and is well placed to contact the peptide. Further clues regarding how CD59 interacts with small peptides arise from the crystal packing of this structure, which shows that a symmetry-related loop comprising residues 20-24 occupies a spatially similar position to the modeled peptide. This higher resolution structure of CD59 will facilitate a more precise dissection of its interactions with C8 and C9 and thus increase the likelihood of designing enhanced CD59-based therapeutics.

Structural features of the ligand binding site on human complement protein C8gamma: a member of the lipocalin family.

Human C8 is one of five components of the cytolytic membrane attack complex of complement. It contains three subunits (C8alpha, C8beta, C8gamma) arranged as a disulfide-linked C8alpha-gamma heterodimer that is noncovalently associated with C8beta. C8gamma has the distinction of being the only lipocalin in the complement system. Lipocalins have a core beta-barrel structure forming a calyx with a binding site for a small hydrophobic ligand. A natural ligand for C8gamma has not been identified; however previous structural studies indicate C8gamma has a typical lipocalin fold that is suggestive of a ligand-binding capability. A distinctive feature of C8gamma is the division of its putative ligand binding pocket into a hydrophilic upper portion and a large hydrophobic lower cavity. Access to the latter is restricted by the close proximity of two tyrosine side chains (Y83 and Y131). In the present study, binding experiments were performed using lauric acid as a pseudoligand to investigate the potential accessibility of the lower cavity. The crystal structure of a C8gamma.laurate complex revealed that Y83 and Y131 can move to allow penetration of the hydrocarbon chain of laurate into the lower cavity. Introducing a Y83W mutation blocked access but had no effect on the ability of C8gamma to enhance C8 cytolytic activity. Together, these results indicate that the lower cavity in C8gamma could accommodate a ligand if such a ligand has a narrow hydrophobic moiety at one end. Entry of that moiety into the lower cavity would require movement of Y83 and Y131, which act as a gate at the cavity entrance.

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.

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.