The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9.

The Membrane Attack Complex (MAC) is responsible for forming large ß-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores.

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and 'locked' C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

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.

Molecular characterization of complement 9 in Epinephelus coioides and differential expression analysis of classical complement genes following Vibrio alginolyticus challenge.

Vibrio alginolyticus is posting an increasing threat to survival of grouper. Classical complement cascade can trigger initiation of immunity, while complement 9 (C9) is a major complement molecule involved in final step of membrane attack complex (MAC) formation. In this study, full-length EcC9 contained an ORF sequence of 1779 bp, encoding a polypeptide of 592 amino acids. A high-level expression of EcC9 mRNA was observed in liver. Following vibrio challenge, increased expression levels of EcC1q, EcBf/C2, EcC4, EcC6, EcC7 and EcC9 mRNA were detected in liver and kidney. These results implied that elevated expression level of classical complement pathway (CCP) and terminal complement components (TCCs) may assess toxicological effect of V. alginolyticus.

Cloning, expression profile of the complement component C9 gene and influence of the recombinant C9 protein on peripheral mononuclear leukocytes transcriptome in half-smooth tongue sole (Cynoglossus semilaevis).

The ninth complement component (C9) is a terminal complement component (TCC) that is involved in creating the membrane attack complex (MAC) on the target cell surface. In this study, the CsC9 (C9 of Cynoglossus semilaevis) cDNA sequence was cloned and characterized. The full-length CsC9 cDNA measured 2,150 bp, containing an open reading frame (ORF) of 1,803 bp, a 5'-untranslated region (UTR) of 24 bp and a 3'-UTR of 323 bp. A domain search revealed that the CsC9 protein contains five domains, including two TSP1s, an LDLRA, an EGF, and a MACPF. Quantitative real-time PCR analysis showed that CsC9 at the mRNA level was expressed in all the tested tissues, with the highest expression being observed in the liver. CsC9 expression is significantly upregulated in the tested tissues after challenge with Vibrio anguillarum. To further characterize the role of CsC9, peripheral blood mononuclear cells of C. semilaevis were used for transcriptome analysis after incubation with recombinant CsC9 (rCsC9) protein. A total of 3,775 significant differentially expressed genes (DEGs) were identified between the control and the rCsC9-treated group, including 2,063 upregulated genes and 1,712 downregulated genes. KEGG analyses revealed that the DEGs were enriched in cell adhesion molecules, cytokine-cytokine receptor interactions, T cell receptor signaling pathways, B cell receptor signaling pathways and Toll-like receptor signaling pathways. The results of this study indicate that in addition to participating in MAC formation, CsC9 might play multiple roles in the innate and adaptive immunity of C. semilaevis.

Molecular characterization, expression analysis, and ontogeny of complement component C9 in southern catfish (Silurus meridionalis).

The complement system plays an important role in host defense against invading microorganisms. Complement component C9 is the last component that is involved in the formation of the membrane attack complex (MAC) on the surface of target cells. In the present study, the full length C9 cDNA sequence of 1984 bp with an open reading frame (ORF) of 1809 bp was cloned from southern catfish (Silurus meridionalis). The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C9 was detected in the liver, spleen, stomach, intestine, and head kidney, with highest levels detected in the liver. The mRNA of C9 was first detected in the yolk syncytial layer at 34 h post fertilization (hpf) with whole mount in situ hybridization, followed by the liver at 36 h post hatching (hph). The mRNA expression of C9 was upregulated significantly in the liver, spleen, and intestine following the injection with Aeromonas hydrophila, suggesting that C9 played an important role in defense against invading pathogens in southern catfish. Therefore, these results provide important information to understand the functions of C9 during fish early development in fish.

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.

The first transmembrane region of complement component-9 acts as a brake on its self-assembly.

Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.

Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation.

The human complement C9 protein (∼65 kDa) is a member of the complement pathway. It plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on the cellular surface of pathogenic bacteria. Here, we charted in detail the structural microheterogeneity of C9 purified from human blood serum, using an integrative workflow combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The proteoform profile of C9 was acquired by high-resolution native mass spectrometry, which revealed the co-occurrence of ∼50 distinct mass spectrometry (MS) signals. Subsequent peptide-centric analysis, through proteolytic digestion of C9 and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) measurements of the resulting peptide mixtures, provided site-specific quantitative profiles of three different types of C9 glycosylation and validation of the native MS data. Our study provides a detailed specification, validation, and quantification of 15 co-occurring C9 proteoforms and the first direct experimental evidence of O-linked glycans in the N-terminal region. Additionally, next to the two known glycosylation sites, a third novel, albeit low abundant, N-glycosylation site on C9 is identified, which surprisingly does not possess the canonical N-glycosylation sequence N-X-S/T. Our data also reveal a binding of up to two Ca2+ ions to C9. Mapping all detected and validated sites of modifications on a structural model of C9, as present in the MAC, hints at their putative roles in pore formation or receptor interactions. The applied methods herein represent a powerful tool for the unbiased in-depth analysis of plasma proteins and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of the pathophysiological state of the patients.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.

Molecular characterization and expression analysis of complement component C9 gene in the whitespotted bambooshark, Chiloscyllium plagiosum.

Complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions. Activation of the terminal pathway of the complement system leads to the assembly of terminal complement complexes (C5b-9), which induces the characteristic complement-mediated cytolysis. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) is identified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum by RACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42% and 43% identity with human and Xenopus C9 respectively. Through sequence alignment and comparative analysis, the CpC9 protein was found well conserved, with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogenetic analysis places it in a clade with C9 orthologs in higher vertebrate and as a sister taxa to the Xenopus. Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much less or even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response, though it remains to be confirmed by further study.

Construction and characterization of recombinant human C9 or C7 linked to single chain Fv directed to CD25.

Complement-dependent cytotoxicity (CDC) is a potent promoter of tumor clearance during monoclonal antibody therapy. Complement activation on antibody-bearing tumor cells results in formation of the membrane attack complex (MAC), which activates cell death. The complement activation cascade that bridges between antibody binding and MAC formation is regulated by complement inhibitors that are over-expressed on tumor cells. In order to bypass those complement regulators, we have designed an immunoconjugate composed of a humanized single chain Fv of an anti-Tac (CD25) monoclonal antibody fused at its C terminus either to complement protein C9 (scFv-C9) or to complement C7 (scFv-C7) and tagged with six histidines at the C terminal end. Recombinant scFv-C9 and scFv-C7 were expressed in 293T cells and purified. Both are shown to efficiently bind to CD25-positive tumor cells. In addition, scFv-C9, but not scFv-C7, increases MAC deposition on the cells and enhances complement-mediated cell death of target CD25-positive cells. Thus, scFv-C9 fusion protein is potentially a novel reagent for application in cancer immunotherapy.

Crystal structure and functional characterization of the complement regulator mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1).

The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin-associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nm and 2.5 nm, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ∼146 Šlong. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9.

[Molecular cloning, sequence analysis and expression of ayu complement component C9 gene].

C9, a component of the membrane attack complex, participates in the final stage of the complement cascade which lyses foreign organisms by disrupting the integrity of their cell membranes. In the present study, a full-length ayu C9 (aC9) cDNA was cloned which contains 2,125 nucleotides and encodes a protein of 592 amino acids. A signal peptide was deposited in the N-terminal 22 residues. The deduced amino acid sequence of aC9 showed 56.8% identity to the C9 of rainbow trout, and 40.9% to 53.8% identity to the C9 of other teleosts. RT-PCR analysis demonstrated that the mRNA of aC9 was expressed in the liver, spleen, intestine, gill and muscle of healthy ayu fish with the highest level in the liver. Quantitative RT-PCR analysis showed that aC9 transcripts were significantly up-regulated in the liver at 4 h post Listonella anguillarum infection, peaked at 16 h post injection. Western blotting analysis revealed that serum aC9 significantly increased in Listonella anguillarum infected ayu fish. Our results suggested that aC9 may play an important role in fish immune response of anti-bacteria.

PRELP protein inhibits the formation of the complement membrane attack complex.

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.

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.

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.

Topology of the membrane-bound form of complement protein C9 probed by glycosylation mapping, anti-peptide antibody binding, and disulfide modification.

The two N-linked oligosaccharides in native human C9 were deleted by site-specific mutagenesis. This aglycosyl-C9 did not differ from its native form in hemolytic and bactericidal activity. A new N-glycosylation site (K311N/E313T) was introduced into the turn of a helix-turn-helix [HTH] fold that had been postulated to form a transmembrane hairpin in membrane-bound C9. This glycosylated form of human C9 was as active as the native protein suggesting that the glycan chain remains on the external side of the membrane and that translocation of this hairpin is not required for membrane anchoring. Furthermore, flow cytometry provided evidence for the recognition of membrane-bound C9 on complement-lysed ghosts by an antibody specific for the HTH fold. A new N-glycosylation site (P26N) was also introduced close to the N-terminus of C9 to test whether this region was involved in C9 polymerization, which is thought to be required for cytolytic activity of C9. Again, this glycosylated C9 was as active as native C9 and could be induced to polymerize by heating or incubation with metal ions. The two C-terminal cystines within the MACPF domain could be eliminated partially or completely without affecting the hemolytic activity. Free sulfhydryl groups of unpaired cysteines in such C9 mutants are blocked since they could not be modified with SH-specific reagents. These results are discussed with respect to a recently proposed model that, on the basis of the MACPF structure in C8alpha, envisions membrane insertion of C9 to resemble the mechanism by which cholesterol-dependent cytolysins enter a membrane.