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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 the 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.

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 >220 M 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.

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP a-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1b cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation. [ABSTRACT FROM AUTHOR]

The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1β, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response. [ABSTRACT FROM AUTHOR]

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1β cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation.

We are currently experiencing an enduring global epidemic of obesity and diabetes. It is now understood that chronic low-grade tissue inflammation plays an important role in metabolic disease, brought upon by increased uptake of a so-called Western diet, and a more sedentary lifestyle. Many evolutionarily conserved links exist between metabolism and the immune system, and an imbalance in this system induced by chronic over-nutrition has been termed 'metaflammation'. The complement system is an important and evolutionarily ancient part of innate immunity, but recent work has revealed that complement not only is involved in the recognition of pathogens and induction of inflammation, but also plays important roles in cellular and tissue homeostasis. Complement can therefore contribute both positively and negatively to metabolic control, depending on the nature and anatomical site of its activity. This review will therefore focus on the interactions of complement with mechanisms and tissues relevant for metabolic control, obesity and diabetes. [ABSTRACT FROM AUTHOR]

Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b‐binding protein C‐terminal‐α‐/β‐chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent‐positive regulator of the AP, the human factor H‐related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VHH targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab‐recognising epitopes [VHH(T) or VHH(P)], respectively, were used as HER2 anchoring moieties. Optimised high‐FHR4 valence heteromultimeric immunoconjugates [FHR4/VHH(T) or FHR4/VHH(P)] were selected by sequential cell cloning and a selective multistep His‐Trap purification. Optimised FHR4‐heteromultimeric immunoconjugates successfully overcame FH‐mediated complement inhibition threshold, causing increased C3b deposition on SK‐OV‐3, BT474 and SK‐BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement‐dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane‐anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement‐dependent cell‐mediated cytotoxicity. We showed that the degree of FHR4‐multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH‐mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady‐state towards activation on tumour cell surface.

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite’s ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts.

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite's ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts. [ABSTRACT FROM AUTHOR]

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation. [ABSTRACT FROM AUTHOR]

Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b‐binding protein C‐terminal‐α‐/β‐chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent‐positive regulator of the AP, the human factor H‐related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VHH targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab‐recognising epitopes [VHH(T) or VHH(P)], respectively, were used as HER2 anchoring moieties. Optimised high‐FHR4 valence heteromultimeric immunoconjugates [FHR4/VHH(T) or FHR4/VHH(P)] were selected by sequential cell cloning and a selective multistep His‐Trap purification. Optimised FHR4‐heteromultimeric immunoconjugates successfully overcame FH‐mediated complement inhibition threshold, causing increased C3b deposition on SK‐OV‐3, BT474 and SK‐BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement‐dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane‐anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement‐dependent cell‐mediated cytotoxicity. We showed that the degree of FHR4‐multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH‐mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady‐state towards activation on tumour cell surface. [ABSTRACT FROM AUTHOR]

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.

Objectives: The rise in multidrug resistant Neisseria gonorrhoeae poses a threat to healthcare, while the development of an effective vaccine has remained elusive due to antigenic and phase variability of surface-expressed proteins. In the current study, we identified a fully conserved surface expressed protein and characterized its suitability as a vaccine antigen.Methods: An in silico approach was used to predict surface-expressed proteins and analyze sequence conservation and phase variability. The most conserved protein and its surface-exposed Loop 2, which was displayed as both a structural and linear epitope on the oligomerization domain of C4b binding protein, were used to immunize mice. Immunogenicity was subsequently analyzed by determination of antibody titers and serum bactericidal activity.Results: MtrE was identified as one of the most conserved surface-expressed proteins. Furthermore, MtrE and both Loop 2-containing fusion proteins elicited high protein-specific antibody titers and particularly the two Loop 2 fusion proteins showed high anti-Loop 2 titers. In addition, antibodies raised against all three proteins were able to recognize MtrE expressed on the surface of N. gonorrhoeae and showed high MtrE-dependent bactericidal activity.Conclusions: Our results show that MtrE and Loop 2 are promising novel conserved surface-expressed antigens for vaccine development against N. gonorrhoeae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Key Clinical Message: In this study, we present the first case of a 34‐year‐old Surinamese female with ischemic retinopathy and increased free protein S due to C4BP deficiency. Possibly, the low PS/C4BP complex level has increased the risk of arterial thrombosis in our patient. [ABSTRACT FROM AUTHOR]

Protein S is a vitamin K-dependent plasma glycoprotein circulating in plasma at a concentration of around 350 nM. Approximately 60% of protein S in human plasma is bound to the complement regulatory protein C4b-binding protein (C4BP) in a highaffinity, high-molecular-weight complex. Protein S in plasma has multiple anticoagulant properties and heterozygous protein S deficiency is associated with increased risk of venous thrombosis. Homozygous deficiency in man and mice is associated with severe thrombosis in fetal life, defects in the vascular system development, and not compatible with life. Protein S has additional functions beyond being an anticoagulant. It affects the complement regulatory properties of C4BP, and moreover, protein S interacts with tyrosine kinase receptors of the TAM family, which comprises Tyro3, Axl, and Mer. The TAM receptor interaction is important for the ability of protein S to stimulate phagocytosis of apoptotic cells. This review will discuss the multiple functions of protein S, describing its role as cofactor to activated protein C with a subsequent focus on the other functions of protein S. [ABSTRACT FROM AUTHOR]

The use of C3d, the final degradation product of complement protein C3, as a “natural” adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF). High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2. These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Inflammasome 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. Methods: Macrophages 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. Results: C4BP 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/interpretation: IAPP 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. [ABSTRACT FROM AUTHOR]

Type 2 Diabetes (T2D) is a disease of increasing importance and represents a growing burden on global healthcare and human health. In T2D, loss of effectiveness of insulin signaling in peripheral tissues cannot be compensated for by adequate insulin secretion, leading to hyperglycemia and resultant complications. In recent years, inflammation has been identified as a central component of T2D, both in inducing peripheral insulin resistance as well as in the pancreatic islet, where it contributes to loss of insulin secretion and death of insulin-secreting beta cells. In this review we will focus on non-traditional roles of complement proteins which have been identified in T2D-associated inflammation, beta cell secretory function, and in maintaining homeostasis of the pancreatic islet. Improved understanding of both traditional and novel roles of complement proteins in T2D may lead to new therapeutic approaches for this global disease. [ABSTRACT FROM AUTHOR]

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig -transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans , but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. [ABSTRACT FROM AUTHOR]

Multivalente Liganden von Todesrezeptoren sind vielversprechende tumorspezifische Wirkstoffe, da sie eine apoptotische Kaskade in Krebszellen induzieren. Hier beschreiben wir einen modularen Ansatz für den Aufbau von Konstrukten, die an den Todesrezeptor 5 (“death receptor 5”, DR5) binden. In diesen Konstrukten sind mehrere Kopien gegen DR5 gerichteter Peptide (DR5TP) kovalent mit biomolekularen Gerüsten verknüpft. Mit dieser Strategie gelingt eine effiziente Oligomerisierung der Peptide in verschiedenen räumlichen Orientierungen durch enzymvermittelte Konjugationen oder rekombinante Produktion. Heptamere Konstrukte mit einem kurzen Gerüst (60 – 75 Reste) der C ‐ terminalen Oligomerisierungdomäne des humanen C4b ‐ Bindeproteins zeigten eine bemerkenswert hohe proapoptotische Aktivität (EC50=3 nm), wenn DR5TP an den Carboxyterminus ligiert wurde. Unsere Daten stützen die Ansicht, dass der Interligandabstand, die relative räumliche Orientierung und die Kopienzahl der rezeptorbindenden Module wesentliche Kriterien für die Rezeptoraktivierung und Zelltötung sind. Gegen den Todesrezeptor 5 (DR5) gerichtete Peptide wurden kovalent mit biomolekularen Gerüsten verknüpft, um multivalente Liganden zu erhalten, die spezifisch eine apoptotische Kaskade in Krebszellen induzieren. Anzahl und räumliche Orientierung der Kopien entscheiden über ihr Vermögen zur Rezeptoraktivierung. [ABSTRACT FROM AUTHOR]

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 n m) 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. [ABSTRACT FROM AUTHOR]

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. In this study, we report that the recombinant proteins LIC10507, LIC10508 and LIC10509 are recognized by confirmed leptospirosis serum samples at both phases of the disease. The recombinant rLIC10508 and rLIC10507 are plasminogen (PLG)-binding proteins, capable of generating plasmin in the presence of a PLG activator. The proteins bind to PLG in a dose-dependent and saturable manner, fulfilling host-ligand interaction. Furthermore, rLIC10508 interacts with fibrinogen (Fg), plasma fibronectin and C4b binding protein (C4BP). The binding of rLIC10508 to Fg decreases the fibrin clotting in a thrombin-catalyzed reaction. The incubation with 4 μM of protein promoted 40% inhibition upon clotting formation. C4BP bound to rLIC10508 retained its cofactor activity for factor I promoting the cleavage of C4b protein, which may reduce the membrane attack complex formation. Although these proteins have high amino acid sequence similarity, rLIC10508 is the most talented of the three, a behavior that might be explained by its unique putative 3D structure, whereas structures of rLIC10507 and rLIC10509 are very similar. Plasmin generation (rLIC10507 and rLIC10508), together with decreasing fibrin clot formation (rLIC10508) and impairment of the complement system (rLIC10508) may help the bacteria to overcome host defense, facilitating the infection process. [ABSTRACT FROM AUTHOR]

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP-/-) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP-/- mice were fertile and no significant differences were observed between the C4BP-/- and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation. [ABSTRACT FROM AUTHOR]

Abstract: Nanofiltration assures that protein therapeutics are free of adventitious agents such as viruses. Nanofilter pores must allow passage of protein drugs but be small enough to retain viruses. Five nanofilters have been evaluated to identify those that can be used interchangeably to yield a high purity Coagulation Factor IX product. When product preparations prior to nanofiltration were analyzed using electrophoresis, Western blot, liquid chromatography – tandem mass spectrometry and size exclusion HPLC, factor IX, inter – α – trypsin inhibitor and C4b binding protein (C4BP) were observed. C4BP was removed from product by all five nanofilters when nanofiltration was performed at physiological ionic strength. However, at high ionic strength, C4BP was removed by only two nanofilters. HPLC indicated that the Stokes radius of C4BP was larger at low ionic strength than at high ionic strength. The results suggest that C4BP exists in an open conformation at physiological ionic strength and is removed by nanofiltration whereas, at high ionic strength, the protein collapses to an extent that allows passage through some nanofilters. Manufacturers should be aware that protein contaminants in other nanofiltered protein drugs could behave similarly and conditions of nanofiltration must be evaluated to ensure consistent product purity. [Copyright &y& Elsevier]

Abstract: Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K D ) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. [Copyright &y& Elsevier]

Abstract: In the Alzheimer''s disease (AD) brain, binding of Clq within the Cl complex, the initiating molecule of the classical complement pathway, to apoptotic cells, DNA and amyloid-β (Aβ), the major constituent of senile plaques, can initiate complement activation. However, the extent of activation is determined by the balance between activation and inhibition. Fluid-phase complement inhibitor C4b-binding protein (C4BP) was immunohistochemically detected in Aβ plaques and on apoptotic cells in AD brain. In vitro, C4BP bound apoptotic and necrotic but not viable brain cells (astrocytes, neurons and oligodendrocytes) and limited complement activation on dead brain cells. C4BP also bound Aβ1–42 peptide directly, via the C4BP α-chain, and limited the extent of complement activation by Aβ. C4BP levels in cerebrospinal fluid (CSF) of dementia patients and controls were low compared to levels in plasma and correlated with CSF levels of other inflammation-related factors. In conclusion, C4BP binds to dead brain cells and Aβ peptide in vitro, is present in CSF and possibly protects against excessive complement activation in AD brains. [Copyright &y& Elsevier]

Abstract: Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n =202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125I-labeled proteins. Blood isolates of GAS (n =10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6–12% for FH and 19–56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n =10), three bound FH (binding level: 8–11%) and two C4BP (36–39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci. [Copyright &y& Elsevier]

Abstract: Complement and dendritic cells (DCs) are essential components of innate immunity. Both participate in local inflammation and moreover have roles in the initiation of the acquired immunity response and in the maintenance of tolerance. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, Factor I, Factor B and complement receptors 3 and 4. In this study, we demonstrate that human DCs are a source of other soluble complement proteins including C1q, C4b binding protein (C4BP), C7 and C8. Complement receptors (CR)1 and the CD18 chain (common for CR3 and CR4) were also present on DCs while CR2 was not detected. [Copyright &y& Elsevier]

Abstract: Many Gram-positive bacteria express surface proteins that bind human plasma proteins. These bacterial proteins, and derivatives of them, are of interest for analysis of bacterial pathogenesis and as immunochemical tools. Well-characterized examples include the IgG-binding reagents staphylococcal protein A and streptococcal protein G, and the recently described streptococcal IgA-binding peptide Sap. Here, we show that a peptide derived from the streptococcal M22 protein can be used for single-step affinity purification of the human complement regulator C4b-binding protein (C4BP). Binding of C4BP was strongly enhanced by dimerization of the peptide via a C-terminal cysteine residue not present in the intact M22 protein. The purified C4BP had the expected binding characteristics, and acted as a cofactor for factor I in the degradation of C4b. Passage of serum through a peptide column under non-saturating conditions resulted in binding of >99.5% of serum C4BP, implying that such a column can be used to deplete serum of C4BP. These data indicate that the C4BP-binding peptide is a versatile tool that can be used for simple and rapid purification of biologically active human C4BP or for removal of C4BP from serum. [Copyright &y& Elsevier]

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2–4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8–11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1–3 of the C4b-binding protein (C4bp-PI) was found to be effective. [Copyright &y& Elsevier]

Complement regulator C4b-binding protein (C4BP) and the anticoagulant vitamin K-dependent protein S form a high affinity complex in human plasma. C4BP is composed of seven α-chains and a unique β-chain, each chain comprising repeating complement control protein (CCP) modules. The binding site for protein S mainly involves the first of the three β-chain CCPs (CCP1). However, recently it has been suggested that CCP2 of the β-chain also contributes to the binding of protein S. To elucidate the structural background for the involvement of CCP2 in the protein S binding, several recombinant β-chain CCP1-2 variants having mutations in CCP2 were expressed and tested for protein S binding. Mutations were chosen based on analysis of a homology model of the β-chain and included R60A/R101A, D66A, L105A, F114A/I116A and H108A. All mutant proteins bound equally well as recombinant wild type to protein S. Several monoclonal antibodies against the β-chain CCP2 were raised and their influence on protein S binding characterized. Taken together, the results suggest that the role of CCP2 in protein S binding is to orient and stabilize CCP1 rather than to be directly part of the binding site. [ABSTRACT FROM AUTHOR]

It appears that consistent changes in the levels of activity of a small cohort of genes (probably less than 1% of all active genes) occur in all mammalian cells during ageing. We have studied this phenomenon in rat liver using an optimised form of differential display. During this investigation we observed one gene which exhibited a decline in expression in livers from young adult (6 months) to aged adult (24 months) animals. The differential expression of this gene was confirmed by single strand conformational polymorphism (SSCP) gel analysis and Northern blotting. Densitometry of the latter indicated that there was a decline of 35% in its expression with age. Characterisation of the isolated PCR fragment demonstrated it to code for the alpha subchain of the complement 4 binding protein (C4BP). The C4BP is a key regulatory protein of the complement system and this observation therefore indicates that a decline in the efficiency of the complement system may be an important factor in the overall decline in immune function that has been observed during ageing. [ABSTRACT FROM AUTHOR]

C4BP has a central role in regulating the classical complement (C′) pathway, but it is still uncertain whether or not it is consumed during in vivo complement activation. Attempts to demonstrate changes in C4BP plasma levels in systemic lupus erythematosus and essential mixed cryoglobulinaemia have failed, probably due to up-regulation of this protein during the inflammatory reaction. We have studied one patient with severe post-transfusion complement-mediated anaphylaxis (CMA), and 67 patients with hereditary C1 inhibitor deficiency (hereditary angioedema (HAE)). The first of these two conditions is characterized by the absence of systemic inflammatory reaction and the second by acute and chronic activation of the C′ classical pathway. C4BP, C4BP–C4b complex, and soluble terminal C′ complex (sC5b-9) were measured in the patients' plasmas by ELISA techniques and C3a and C4a by radioimmunoassays. In CMA, 15 min after the transfusion, there was a massive C′ activation, with increases in C4a, C3a, sC5b-9, C4BP–C4b complexes and decreases in C4, C3 and C4BP. All parameters reverted to preinfusion values within 24 h. Depletion of C4 was correlated with that of C4BP. In patients with HAE, the median value of C4BP (83% range 54–165) was significantly lower (P < 0.0001) than in normal controls (99% range 70–159), with no difference between patients in remission or during acute attacks. C4BP–C4b complexes could not be detected in HAE patients. The results of this study indicate that C4BP is consumed in vivo during acute, and possibly during chronic activation of the C′ classical pathway, and that this protein, after interaction with C4b, not longer circulates in plasma. [ABSTRACT FROM AUTHOR]

We investigated the serial changes in blood protein S (PS) and related proteins in 11 normal pregnant women. The PS activity decreased significantly in the third trimester and reached minimum levels (23.3%) one hour after delivery. Although the PS activity was reduced markedly below the normal limits, all the women delivered safely. The mechanisms that cause the reduction in PS activity and the clinically dangerous conditions involving PS activity during pregnancy warrant further investigation. [ABSTRACT FROM AUTHOR]