Complement Regulator C4b-binding Protein Research Articles

Survival of Borrelia burgdorferi Strain B31 in Human Serum Is Not Dependent on C4BP Binding to the Bacterial Surface.

Lyme disease is a vector-borne illness caused by spirochetes belonging to the Borrelia burgdorferi species group. These bacteria employ several mechanisms to survive within the vertebrate host, including evasion of the complement system. In this study, we examine the protection against human serum killing by the binding of host complement regulators C4b-binding protein (C4BP) and factor H (FH) to the bacterial surface of B. burgdorferi. Via serum depletion of isolated complement regulators, we found that the absence of C4BP did not alter the survival of B. burgdorferi strain B31; however, the removal of FH increased the sensitivity of this strain to human serum as previously described. The B. garinii seabird-isolated strain Far04, on the other hand, did not bind any complement regulators of human origin and was serum-sensitive, indicating its special host species specificity.

The Hidden Side of Complement Regulator C4BP: Dissection and Evaluation of Its Immunomodulatory Activity.

C4b-binding protein (C4BP) is a well-known regulator of the complement system that holds additional and important activities unrelated to complement inhibition. Recently, we have described a novel immunomodulatory activity in the minor C4BP(β-) isoform directly acting over inflammatory phagocytes. Here we show that incorporation of the β-chain to the C4BP α-chain oligomer interferes with this immunomodulatory activity of C4BP. Moreover, an oligomeric form including only the complement control protein 6 (CCP6) domain of the C4BP α-chain (PRP6-HO7) is sufficient to “reprogram” monocyte-derived DCs (Mo-DCs) from a pro-inflammatory and immunogenic phenotype to an anti-inflammatory and tolerogenic state. PRP6-HO7 lacks complement regulatory activity but retains full immunomodulatory activity over inflammatory Mo-DCs induced by TLRs, characterized by downregulation of relevant surface markers such as CD83, HLA-DR, co-stimulatory molecules such as CD86, CD80 and CD40, and pro-inflammatory cytokines such as IL-12 and TNF-α. Furthermore, PRP6-HO7-treated Mo-DCs shows increased endocytosis, significantly reduced CCR7 expression and CCL21-mediated chemotaxis, and prevents T cell alloproliferation. Finally, PRP6-HO7 shows also full immunomodulatory activity over Mo-DCs isolated from lupus nephritis patients with active disease, even without further pro-inflammatory stimulation. Therefore PRP6-HO7, retaining the immunomodulatory activity of C4BP(β-) and lacking its complement regulatory activity, might represent a promising and novel alternative to treat autoimmune diseases.

Complement evasion by the human respiratory tract pathogens Haemophilusinfluenzae and Moraxellacatarrhalis.

All infective bacterial species need to conquer the innate immune system in order to colonize and survive in their hosts. The human respiratory pathogens Haemophilusinfluenzae and Moraxellacatarrhalis are no exceptions and have developed sophisticated mechanisms to evade complement-mediated killing. Both bacterial species carry lipooligosaccharides preventing complement attacks and attract and utilize host complement regulators C4b binding protein and factor H to inhibit the classical and alternative pathways of complement activation, respectively. In addition, the regulator of the terminal pathway of complement activation, vitronectin, is hijacked by both bacteria. An array of different outer membrane proteins (OMP) in H.influenzae and M.catarrhalis simultaneously binds complement regulators, but also plasminogen. Several of the bacterial complement-binding proteins are important adhesins and contain highly conserved regions for interactions with the host. Thus, some of the OMP are viable targets for new therapeutics, including vaccines aimed at preventing respiratory tract diseases such as otitis media in children and exacerbations in patients suffering from chronic obstructive pulmonary disease.

The multifaceted roles of Leptospira enolase

A previous study had demonstrated that Leptospira enolase is secreted extracellularly by a yet unknown mechanism and reassociates with the bacterial membrane. Surface-anchored leptospiral enolase displays plasminogen binding activity. In this work, we explored the consequences of this interaction and also assessed whether Leptospira enolase might display additional moonlighting functions by interacting with other host effector proteins. We first demonstrated that enolase-bound plasminogen is converted to its active form, plasmin. The protease plasmin targets human fibrinogen and vitronectin, but not the complement proteins C3b and C5. Leptospira enolase also acts as an immune evasion protein by interacting with the negative complement regulators C4b binding protein and factor H. Once bound to enolase, both regulators remain functional as cofactors of factor I, mediating cleavage of C4b and C3b. In conclusion, enolase may facilitate leptospiral survival and dissemination, thus contributing to bacterial virulence. The identification and characterization of moonlighting proteins is a growing field of bacterial pathogenesis, as these multifaceted proteins may represent potential future therapeutic targets to fight bacterial infections.

The complement regulator C4b-binding protein inhibits islet amyloid polypeptide-induced inflammasome activation

The complement regulator C4b-binding protein inhibits islet amyloid polypeptide-induced inflammasome activation

Moraxella catarrhalis Binds Plasminogen To Evade Host Innate Immunity.

Several bacterial species recruit the complement regulators C4b-binding protein, factor H, and vitronectin, resulting in resistance against the bactericidal activity of human serum. It was recently demonstrated that bacteria also bind plasminogen, which is converted to plasmin that degrades C3b and C5. In this study, we found that a series of clinical isolates (n = 58) of the respiratory pathogen Moraxella catarrhalis, which is commonly isolated from preschool children and adults with chronic obstructive pulmonary disease (COPD), significantly binds human plasminogen. Ubiquitous surface protein A2 (UspA2) and hybrid UspA2 (UspA2H) were identified as the plasminogen-binding factors in the outer membrane proteome of Moraxella. Furthermore, expression of a series of truncated recombinant UspA2 and UspA2H proteins followed by a detailed analysis of protein-protein interactions suggested that the N-terminal head domains bound to the kringle domains of plasminogen. The binding affinity constant (KD) values of full-length UspA2(30-539) (amino acids 30 to 539 of UspA2) and full-length UspA2H(50-720) for immobilized plasminogen were 4.8 × 10(-8) M and 3.13 × 10(-8) M, respectively, as measured by biolayer interferometry. Plasminogen bound to intact M. catarrhalis or to recombinant UspA2/UspA2H was readily accessible for a urokinase plasminogen activator that converted the zymogen into active plasmin, as verified by the specific substrate S-2251 and a degradation assay with fibrinogen. Importantly, plasmin bound at the bacterial surface also degraded C3b and C5, which consequently may contribute to reduced bacterial killing. Our findings suggest that binding of plasminogen to M. catarrhalis may lead to increased virulence and, hence, more efficient colonization of the host.

The α7β0 Isoform of the Complement Regulator C4b-Binding Protein Induces a Semimature, Anti-Inflammatory State in Dendritic Cells

The classical pathway complement regulator C4b-binding protein (C4BP) is composed of two polypeptides (α- and β-chains), which form three plasma oligomers with different subunit compositions (α7β1, α7β0, and α6β1). We show in this article that the C4BP α7β0 isoform (hereafter called C4BP[β(-)] [C4BP lacking the β-chain]), overexpressed under acute-phase conditions, induces a semimature, tolerogenic state on human monocyte-derived dendritic cells (DCs) activated by a proinflammatory stimulus. C4BP isoforms containing β-chain (α7β1 and α6β1; C4BP[β(+)]) neither interfered with the normal maturation of DCs nor competed with C4BP(β(-)) activity on these cells. Immature DCs (iDCs) treated with C4BP(β(-)) retained high endocytic activity, but, upon LPS treatment, they did not upregulate surface expression of CD83, CD80, and CD86. Transcriptional profiling of these semimature DCs revealed that treatment with C4BP(β(-)) prevented the induction of IDO and BIC-1, whereas TGF-β1 expression was maintained to the level of iDCs. C4BP(β(-))-treated DCs were also unable to release proinflammatory Th1 cytokines (IL-12, TNF-α, IFN-γ, IL-6, IL-8) and, conversely, increased IL-10 secretion. They prevented surface CCR7 overexpression and, accordingly, displayed reduced chemotaxis, being morphologically indistinguishable from iDCs. Moreover, C4BP(β(-))-treated DCs failed to enhance allogeneic T cell proliferation, impairing IFN-γ production in these cells and, conversely, promoting CD4(+)CD127(low/neg)CD25(high)Foxp3(+) T cells. Deletion mutant analysis revealed that the complement control protein-6 domain of the α-chain is necessary for the tolerogenic activity of C4BP(β(-)). Our data demonstrate a novel anti-inflammatory and immunomodulatory function of the complement regulator C4BP, suggesting a relevant role of the acute-phase C4BP(β(-)) isoform in a number of pathophysiological conditions and potential applications in autoimmunity and transplantation.

Complement regulator C4BP binds to Staphylococcus aureus surface proteins SdrE and Bbp inhibiting bacterial opsonization and killing

Staphylococcus aureus is a premier human pathogen and the most common cause of osteoarticular, wound, and implanted device infections. We recently demonstrated S. aureus efficiently binds the classical complement regulator C4b-binding protein (C4BP) inhibiting antibody-initiated complement-mediated opsonization. Here we identify S. aureus surface protein SdrE as a C4BP-binding protein. Recombinant SdrE and recombinant bone sialoprotein-binding protein (Bbp), an allelic variant of SdrE, both efficiently bound to C4BP in heat-inactivated human serum. We previously described SdrE as binding alternative pathway regulator factor H. Recombinant SdrE and Bbp efficiently bound C4BP and factor H in serum without apparent interference. Gain of function studies utilizing Lactococcus lactis clones expressing SdrE or Bbp increased serum C4BP and factor H binding, compared with empty-vector control (WT) approximately 2-fold. Correspondingly, classical pathway-mediated C3-fragment opsonization and bacterial killing by human neutrophils decreased by half for L. lactis clones expressing SdrE or Bbp compared with WT. In summary, we identify SdrE and allelic variant Bbp as S. aureus surface proteins that bind the complement regulator C4BP inhibiting classical pathway-mediated bacterial opsonization and killing.

Complement regulator C4BP binds to Staphylococcus aureus and decreases opsonization

Staphylococcus aureus is the major cause of human skin and soft-tissue infections as well as invasive infections like post-operative wound infections, septic arthritis, and osteomyelitis. The complement system plays an important role in the immunological control of many bacteria, but can be inhibited by a variety of strategies including recruitment of complement regulatory proteins like C4b-binding protein (C4BP). These experiments demonstrate that S. aureus opsonization with C4b occurs rapidly in serum and is predominantly initiated by anti-staphylococcal antibodies. Much of the S. aureus-bound C4b is quickly cleaved to the inactive forms iC4b and C4d. Clinical S. aureus strains rapidly bind significant amounts of the complement regulator C4BP from serum. S. aureus also binds purified C4BP. S. aureus-bound C4BP functions as a cofactor for factor I-mediated C4b cleavage to iC4b and C4d. In the absence of factor I, C4BP decreases classical pathway-mediated deposition of C3b on the S. aureus surface by inhibiting the classical pathway C3-convertase. In summary, C4BP is recruited to the S. aureus surface where it functions to inhibit C4 complement effectors, suggesting a previously undescribed immune evasion strategy for this pathogen.

Human Pentraxin 3 Binds to the Complement Regulator C4b-Binding Protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1–3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

C4b-Binding Protein: A Forgotten Factor in Thrombosis and Hemostasis

Blood coagulation and complement pathways are two important natural defense systems. The high affinity interaction between the anticoagulant vitamin K-dependent protein S and the complement regulator C4b-binding protein (C4BP) is a direct physical link between the two systems. In human plasma, ~70% of total protein S circulates in complex with C4BP; the remaining is free. The anticoagulant activity of protein S is mainly expressed by the free form, although the protein S-C4BP complex has recently been shown to have some anticoagulant activity. The high affinity binding of protein S to C4BP provides C4BP with the ability to bind to negatively charged phospholipid membranes, which serves the purpose of localizing complement regulatory activity close to the membrane. Even though C4BP does not directly affect the coagulation system, it still influences the regulation of blood coagulation through its interaction with protein S. This is particularly important in states of inherited deficiency of protein S where the tight binding of protein S to C4BP results in a pronounced and selective drop in concentration of free protein S, whereas the concentration of protein S in complex with C4BP remains relatively unchanged. This review summarizes the current knowledge on C4BP with respect to its association with thrombosis and hemostasis.

Binding of complement regulator C4b binding protein to Flavivirus Nonstructural Protein NS1 modulates its function in solution and on the cell surface (154.22)

Abstract The complement system plays an important role in the innate immune response to many pathogens including Flaviviruses. Flavivirus NS1 is a secreted glycoprotein that is absent from viral particles but accumulates at high levels in the blood of infected patients where it may also bind back to the cell surface. Our previous work defined an immune evasion role of Flavivirus NS1 in limiting classical/lectin pathway activation by forming a complex with C1s and C4 to promote the cleavage of C4 to C4b. Herein, we demonstrate a second mechanism by which NS1 antagonizes complement activation. In this case, dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 directly associate with C4b binding protein (C4BP), a plasma complement regulatory protein that attenuates the classical/lectin pathway. C4BP is a cofactor for the serine protease factor I to cleave C4b and thereby inhibits classical/lectin pathway C3 convertase (C4b2a) activity. Specifically, binding of NS1 to C4BP results in more efficient factor I-mediated degradation of C4b by C4BP in solution. Furthermore, NS1 recruits C4BP to the surface of cells to inactivate C4b on the plasma membrane. These studies define immune evasion mechanisms by which NS1 reduces C4’s activating capacity. They point to the classical/lectin pathway activation being critical in host defense against Flavivirus infections.

Contribution of Chondroitin Sulfate A to the Binding of Complement Proteins to Activated Platelets

BackgroundExposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.Principal FindingsHuman blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.ConclusionsThis study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases.

Human Complement Regulators C4b-Binding Protein and C1 Esterase Inhibitor Interact with a Novel Outer Surface Protein of Borrelia recurrentis

The spirochete Borrelia recurrentis is the causal agent of louse-borne relapsing fever and is transmitted to humans by the infected body louse Pediculus humanus. We have recently demonstrated that the B. recurrentis surface receptor, HcpA, specifically binds factor H, the regulator of the alternative pathway of complement activation, thereby inhibiting complement mediated bacteriolysis. Here, we show that B. recurrentis spirochetes express another potential outer membrane lipoprotein, termed CihC, and acquire C4b-binding protein (C4bp) and human C1 esterase inhibitor (C1-Inh), the major inhibitors of the classical and lectin pathway of complement activation. A highly homologous receptor for C4bp was also found in the African tick-borne relapsing fever spirochete B. duttonii. Upon its binding to B. recurrentis or recombinant CihC, C4bp retains its functional potential, i.e. facilitating the factor I-mediated degradation of C4b. The additional finding that ectopic expression of CihC in serum sensitive B. burgdorferi significantly increased spirochetal resistance against human complement suggests this receptor to substantially contribute, together with other known strategies, to immune evasion of B. recurrentis.

Functional Characterization of LcpA, a Surface-Exposed Protein ofLeptospiraspp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Borrelia recurrentis interferes with human innate immune defenses through acquisition of complement regulators CFH, CFHR-1, C4b-binding protein and C1-INH

Borrelia recurrentis interferes with human innate immune defenses through acquisition of complement regulators CFH, CFHR-1, C4b-binding protein and C1-INH

The Complement Regulator C4b-Binding Protein (C4BP) Interacts with both the C4c and C4dg Subfragments of the Parent C4b Ligand: Evidence for Synergy in C4BP Subsite Binding,

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionThe Complement Regulator C4b-Binding Protein (C4BP) Interacts with both the C4c and C4dg Subfragments of the Parent C4b Ligand: Evidence for Synergy in C4BP Subsite Binding,Elisa Leung, Anna M. Blom, Liliana Clemenza, and David E. IsenmanCite this: Biochemistry 2007, 46, 11, 3594Publication Date (Web):February 27, 2007Publication History Published online27 February 2007Published inissue 1 March 2007https://doi.org/10.1021/bi700248qCopyright © 2007 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views204Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (40 KB) Get e-Alerts Get e-Alerts

Yersinia enterocolitica serum resistance proteins YadA and Ail are involved in binding the complement regulator C4b-binding protein

Yersinia enterocolitica serum resistance proteins YadA and Ail are involved in binding the complement regulator C4b-binding protein

The Complement Regulator C4b-Binding Protein (C4BP) Interacts with both the C4c and C4dg Subfragments of the Parent C4b Ligand: Evidence for Synergy in C4BP Subsite Binding

C4b-binding protein (C4BP) is a multimeric serum protein that is a potent regulator of the classical and lectin complement pathways. The binding site for C4b has been localized to complement control protein (CCP) domains 1-3 of the C4BP alpha-chain and, in particular, to a cluster of positively charged amino acids predicted to be at the interface between CCP 1 and CCP 2. To determine the regions of C4b contributing to C4BP binding, we have examined via surface plasmon resonance technology the binding of the C4c and C4dg subfragments of C4b to C4BP. At half-physiologic ionic strength, specific and saturable binding was observed for both C4c and C4dg. C4c exhibited much greater ionic strength sensitivity in its binding than did C4dg. Analysis of the effect on binding of the subfragments to various C4b-binding-defective C4BP mutants, together with cross-competition experiments, suggests that the subsites in C4BP for C4c and C4dg are adjacent, but distinct. Additionally, we observed synergy in subsite filling such that the presence of C4dg enhanced the extent of C4c binding over its basal level, and vice versa. The enhanced binding of C4c in the presence of C4dg was not due to an increase in affinity but rather reflected a 2-3-fold increase in the number of sites capable of binding C4c. This suggests the existence of a conformational equilibrium between high- and low-affinity states in the C4c binding subsite within each C4BP subunit, an equilibrium which is shifted in favor of the high-affinity state by the filling of the C4dg subsite.

Extreme Sequence Divergence but Conserved Ligand-Binding Specificity in Streptococcus pyogenes M Protein

Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR) of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP), a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.