Human C4b-binding Protein Research Articles

Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP-binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP, and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to identification of amino acids that are crucial for C4BP-binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Higher-order structure and proteoforms of co-occurring C4b-binding protein assemblies in human serum

The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of two distinct subunits, C4BPα and C4BPβ. These associate with vitamin K-dependent protein S (ProS) forming an ensemble of co-occurring higher-order structures. Here, we characterize these C4BP assemblies. We resolve and quantify isoforms of purified human serum C4BP using distinct single-particle detection techniques: charge detection mass spectrometry, and mass photometry accompanied by high-speed atomic force microscopy. Combining cross-linking mass spectrometry, glycoproteomics, and structural modeling, we report comprehensive glycoproteoform profiles and full-length structural models of the endogenous C4BP assemblies, expanding knowledge of this key complement inhibitor’s structure and composition. Finally, we reveal that an increased C4BPα to C4BPβ ratio coincides with elevated C-reactive protein levels in patient plasma samples. This observation highlights C4BP isoform variation and affirms a distinct role of co-occurring C4BP assemblies upon acute phase inflammation.

Diverse Functions of C4b-Binding Protein in Health and Disease.

C4b-binding protein (C4BP) is a fluid-phase complement inhibitor that prevents uncontrolled activation of the classical and lectin complement pathways. As a complement inhibitor, C4BP also promotes apoptotic cell death and is hijacked by microbes and tumors for complement evasion. Although initially characterized for its role in complement inhibition, there is an emerging recognition that C4BP functions in a complement-independent manner to promote cell survival, protect against autoimmune damage, and modulate the virulence of microbial pathogens. In this Brief Review, we summarize the structure and functions of human C4BP, with a special focus on activities that extend beyond the canonical role of C4BP in complement inhibition.

A conserved 3D pattern in a Streptococcus pyogenes M protein immunogen elicits M-type crossreactivity

Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens because of type specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was shown to elicit M-type crossreactivity. The basis for this crossreactivity is unknown but may be due in part to antibody recognition of a 3D pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit crossreactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S.pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum-recognized M proteins displayed natively on the strep A surface and promoted the opsonophagocytic killing of strep A strains expressing these M proteins. As C4BP binding is a conserved virulence trait of strep A, we propose that targeting the 3D pattern may prove advantageous in vaccine design.

Interactions of Candida tropicalis pH-related antigen 1 with complement proteins C3, C3b, factor-H, C4BP and complement evasion

Candida, as a part of the human microbiota, can cause opportunistic infections that are either localised or systemic candidiasis. Emerging resistance to the standard antifungal drugs is associated with increased mortality rate due to invasive Candida infections, particularly in immunocompromised patients. While there are several species of Candida, an increasing number of Candida tropicalis isolates have been recently reported from patients with invasive candidiasis or inflammatory bowel diseases. In order to establish infections, C. tropicalis has to adopt several strategies to escape the host immune attack. Understanding the immune evasion strategies is of great importance as these can be exploited as novel therapeutic targets. C. albicans pH-related antigen 1 (CaPra1), a surface bound and secretory protein, has been found to interact strongly with the immune system and help in complement evasion. However, the role of C. tropicalis Pra1 (CtPra1) and its interaction with the complement is not studied yet. Thus, we characterised how pH-related antigen 1 of C. tropicalis (CtPra1) interacts with some of the key complement proteins of the innate immune system. CtPra1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. Recombinant CtPra1, was found to bind human C3 and C3b, central molecules of the complement pathways that are important components of the innate immune system. It was also found to bind human complement regulatory proteins factor-H and C4b-binding protein (C4BP). CtPra1-factor-H and CtPra1-C4BP interactions were found to be ionic in nature as the binding intensity affected by high sodium chloride concentrations. CtPra1 inhibited functional complement activation with different effects on classical (∼20 %), lectin (∼25 %) and alternative (∼30 %) pathways. qPCR experiments using C. tropicalis clinical isolates (oral, blood and peritoneal fluid) revealed relatively higher levels of expression of CtPra1 gene when compared to the reference strain. Native CtPra1 was found to be expressed both as membrane-bound and secretory forms in the clinical isolates. Thus, C. tropicalis appears to be a master of immune evasion by using Pra1 protein. Further investigation using in-vivo models will help ascertain if these proteins can be novel therapeutic targets.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

The Molecular Basis of Human IgG-Mediated Enhancement of C4b-Binding Protein Recruitment to Group A Streptococcus

Streptococcus pyogenes infects over 700 million people worldwide annually. Immune evasion strategies employed by the bacteria include binding of the complement inhibitors, C4b-binding protein (C4BP) and Factor H in a human-specific manner. We recently showed that human IgG increased C4BP binding to the bacterial surface, which promoted streptococcal immune evasion and increased mortality in mice. We sought to identify how IgG promotes C4BP binding to Protein H, a member of the M protein family. Dimerization of Protein H is pivotal for enhanced binding to human C4BP. First, we illustrated that Protein H, IgG, and C4BP formed a tripartite complex. Second, surface plasmon resonance revealed that Protein H binds IgG solely through Fc, but not Fab domains, and with high affinity (IgG-Protein H: KD = 0.4 nM; IgG-Fc-Protein H: KD ≤ 1.6 nM). Each IgG binds two Protein H molecules, while up to six molecules of Protein H bind one C4BP molecule. Third, interrupting Protein H dimerization either by raising temperature to 41°C or with a synthetic peptide prevented IgG-Protein H interactions. IgG-Fc fragments or monoclonal human IgG permitted maximal C4BP binding when used at concentrations from 0.1 to 10 mg/ml. In contrast, pooled human IgG enhanced C4BP binding at concentrations up to 1 mg/ml; decreased C4BP binding at 10 mg/ml occurred probably because of Fab-streptococcal interactions at these high IgG concentrations. Taken together, our data show how S. pyogenes exploits human IgG to evade complement and enhance its virulence. Elucidation of this mechanism could aid design of new therapeutics against S. pyogenes.

Potential Use of Genetically Engineered Variable Lymphocyte Receptor B Specific to Avian Influenza Virus H9N2.

The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2-specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine-serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.

Chimeric proteins containing MAP-1 and functional domains of C4b-binding protein reveal strong complement inhibitory capacities

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1-5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1-5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1-5 was five times more effective than rMAP-1 and rC4BP1-5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities.

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1−5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1−5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1−5 was five times more effective than rMAP-1 and rC4BP1−5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV)

Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

The human serum protein C4b-binding protein inhibits pancreatic IAPP-induced inflammasome activation

Aims/hypothesisInflammasome activation and subsequent IL-1β production is a driver of islet pathology in type 2 diabetes. Oligomers, but not mature amyloid fibrils, of human islet amyloid polypeptide (IAPP), which is co-secreted with insulin, trigger NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome activation. C4b-binding protein (C4BP), present in serum, binds to IAPP and affects transition of IAPP monomers and oligomers to amyloid fibrils. We therefore hypothesised that C4BP inhibits IAPP-mediated inflammasome activation and IL-1β production.MethodsMacrophages were exposed to IAPP in the presence or absence of plasma-purified human C4BP, and inflammasome activation was assessed by IL-1β secretion as detected by ELISA and reporter cell lines. IAPP fibrillation was assessed by thioflavin T assay. Uptake of IAPP–C4BP complexes and their effects on phagolysosomal stability were assessed by flow cytometry and confocal microscopy. The effect of C4BP regulation of IAPP-mediated inflammasome activation on beta cell function was assessed using a clonal rat beta cell line. Immunohistochemistry was used to examine the association of IAPP amyloid deposits and macrophage infiltration in isolated human and mouse pancreatic islets, and expression of C4BP from isolated human pancreatic islets was assessed by quantitative PCR, immunohistochemistry and western blot.ResultsC4BP significantly inhibited IAPP-mediated IL-1β secretion from primed macrophages at physiological concentrations in a dose-dependent manner. C4BP bound to and was internalised together with IAPP. C4BP did not affect IAPP uptake into phagolysosomal compartments, although it did inhibit its formation into amyloid fibrils. The loss of macrophage phagolysosomal integrity induced by IAPP incubation was inhibited by co-incubation with C4BP. Supernatant fractions from macrophages activated with IAPP inhibited both insulin secretion and viability of clonal beta cells in an IL-1β-dependent manner but the presence of C4BP during macrophage IAPP incubation rescued beta cell function and viability. In human and mouse islets, the presence of amyloid deposits correlated with higher numbers of infiltrating macrophages. Isolated human islets expressed and secreted C4BP, which increased with addition of IL-1β.Conclusions/interpretationIAPP deposition is associated with inflammatory cell infiltrates in pancreatic islets. C4BP blocks IAPP-induced inflammasome activation by preventing the loss of macrophage phagolysosomal integrity required for NLRP3 activation. The consequence of this is the preservation of beta cell function and viability. C4BP is secreted directly from human pancreatic islets and this increases in response to inflammatory cytokines. We therefore propose that C4BP acts as an extracellular chaperone protein that limits the proinflammatory effects of IAPP.

Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through structure determination of four sequence-diverse M proteins in complex with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies targeting the M-C4BP interaction, and also inform a path towards vaccine design.

An Apoptosis-Inducing Peptidic Heptad That Efficiently Clusters Death Receptor 5.

Multivalent ligands of death receptors hold particular promise as tumor cell-specific therapeutic agents because they induce an apoptotic cascade in cancerous cells. Herein, we present a modular approach to generate death receptor 5 (DR5) binding constructs comprising multiple copies of DR5 targeting peptide (DR5TP) covalently bound to biomolecular scaffolds of peptidic nature. This strategy allows for efficient oligomerization of synthetic DR5TP-derived peptides in different spatial orientations using a set of enzyme-promoted conjugations or recombinant production. Heptameric constructs based on a short (60-75 residues) scaffold of a C-terminal oligomerization domain of human C4b binding protein showed remarkable proapoptotic activity (EC50=3 nm) when DR5TP was ligated to its carboxy terminus. Our data support the notion that inter-ligand distance, relative spatial orientation and copy number of receptor-binding modules are key prerequisites for receptor activation and cell killing.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.

Arranged Sevenfold: Structural Insights into the C-Terminal Oligomerization Domain of Human C4b-Binding Protein

The complement system as a major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demands a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.

Solid-state NMR sequential assignments of the C-terminal oligomerization domain of human C4b-binding protein

The complement 4 binding protein (C4bp) plays a crucial role in the inhibition of the complement cascade. It has an extraordinary seven-arm octopus-like structure with 7 tentacle-like identical chains, held together at their C-terminal end. The C-terminal domain does oligomerize in isolation, and is necessary and sufficient to oligomerize full-length C4bp. It is predicted to form a seven-helix coiled coil, and its multimerization properties make it a promising vaccine adjuvant, probably by enhancing the structural stability and binding affinity of the presented antigen. Here, we present the solid-state NMR resonance assignment of the human C4bp C-terminal oligomerization Domain, hC4pbOD, and the corresponding secondary chemical shifts.

Enolase ofStreptococcus pneumoniaeBinds Human Complement Inhibitor C4b-Binding Protein and Contributes to Complement Evasion

Streptococcus pneumoniae (pneumococcus) is a pathogen that causes severe local and life-threatening invasive diseases, which are associated with high mortality rates. Pneumococci have evolved several strategies to evade the host immune system, including complement to disseminate and to survive in various host niches. Thus, pneumococci bind complement inhibitors such as C4b-binding protein (C4BP) and factor H via pneumococcal surface protein C, thereby inhibiting the classical and alternative complement pathways. In this study, we identified the pneumococcal glycolytic enzyme enolase, a nonclassical cell surface and plasminogen-binding protein, as an additional pneumococcal C4BP-binding protein. Furthermore, we demonstrated that human, but not mouse, C4BP bound pneumococci. Recombinant enolase bound in a dose-dependent manner C4BP purified from plasma, and the interaction was reduced by increasing ionic strength. Enolase recruited C4BP and plasminogen, but not factor H, from human serum. Moreover, C4BP and plasminogen bound to different domains of enolase as they did not compete for the interaction with enolase. In direct binding assays with recombinant C4BP mutants lacking individual domains, two binding sites for enolase were identified on the complement control protein (CCP) domain 1/CCP2 and CCP8 of the C4BP α-chains. C4BP bound to the enolase retained its cofactor activity as determined by C4b degradation. Furthermore, in the presence of exogenously added enolase, an increased C4BP binding to and subsequently decreased C3b deposition on pneumococci was observed. Taken together, pneumococci specifically interact with human C4BP via enolase, which represents an additional mechanism of human complement control by this versatile pathogen.

Functional Recruitment of Human Complement Inhibitor C4b-Binding Protein to Outer Membrane Protein Rck of Salmonella

Resistance to complement mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 17 kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade mammalian cells. Having recently shown that Rck binds the inhibitor of the alternative pathway of complement, factor H (fH), we hypothesized that Rck can also bind the inhibitor of the classical and lectin pathways, C4b-binding protein (C4BP). Using flow cytometry and direct binding assays, we demonstrate that E. coli expressing Rck binds C4BP from heat-inactivated serum and by using the purified protein. No binding was detected in the absence of Rck expression. C4BP bound to Rck is functional, as we observed factor I-mediated cleavage of C4b in cofactor assays. In competition assays, binding of radiolabeled C4BP to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly non-ionic interactions. Reduced binding of C4BP mutants lacking complement control protein domains (CCPs) 7 or 8 was observed compared to wt C4BP, suggesting that these CCPs are involved in Rck binding. While these findings are restricted to Rck expression in E. coli, these data suggest that C4BP binding may be an additional mechanism of Rck-mediated complement resistance.