Diverse functions of C4b-binding protein in health and disease

Abstract

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. Though 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.

Introduction

Complement is a system of proteins that aid or “complement” immune clearance of pathogens. Complement components are primarily synthesized by hepatocytes and secreted into circulation, but some components are also synthesized locally in tissues by immune cells, endothelial cells, and epithelial cells (1). Complement is activated through three distinct pathways (reviewed in (2)): the classical pathway, the mannose-binding lectin (MBL) pathway, and the alternative pathway. Regardless of the pathway of activation, each converges on the formation of the C3 convertase, an enzyme that cleaves C3 and C5 to produce effector molecules of complement.

Complement activation serves three primary functions in the control of infection. The first is opsonization, in which complement components coat the surface of the pathogen and tag it for recognition by phagocytes via their cognate complement receptors. Second is the production of the anaphylatoxins C3a and C5a, which creates a local inflammatory response that recruits leukocytes to the site of infection and promotes leukocyte activation via their cognate receptors. Third is the assembly of terminal complement components C5b-C9, which insert into lipid bilayers to form a cytolytic pore called the membrane attack complex (MAC) (3, 4). Direct bacteriolysis via MAC is a major method of complement control for Gram-negative bacteria and viruses (5, 6). On the other hand, Gram-positive bacteria, as well as fungi, are relatively resistant to direct cytolysis via a thick cell wall, and complement-mediated control of these organisms is primarily through opsonization and chemoattraction of leukocytes, which support phagocytic clearance (7–9).

Tightly regulated complement activation is important for human health and fitness. The complement cascade is intrinsically restrained by requiring the sequential cleavage of inactive precursors to generate effector molecules. As a second layer of control, membrane-bound and soluble proteinaceous complement inhibitors protect host cells from uncontrolled activation and subsequent damage.

C4b-binding protein (C4BP) is a prominent soluble regulator of the classical and mannose-binding lectin (MBL) pathways of complement activation. Beyond its canonical function in complement inhibition, C4BP has significant roles in other realms of human biology, some of which are complement-independent. In this Brief Review, we will summarize our current understanding and identify future areas for investigation for five roles of C4BP: 1) complement inhibition, 2) microbial complement resistance, 3) complement-independent modulation of microbial pathogenesis, 4) regulation of cell clearance and survival, and 5) control of excessive inflammation in cancer and chronic disease.

Complement inhibition by C4BP

Human C4BP is a glycoprotein complex present abundantly (~200 μg/mL) in healthy human serum (10). C4BP is mainly synthesized in the liver where it is secreted by hepatocytes into the bloodstream, but is also expressed in pancreatic islet cells and lung alveolar cells (11–13). While genetic deficiencies in some complement components have been described, there are no reported human deficiencies in C4BP, implying its importance to human biology (14).

C4BP exists as a multimer of C4BP α chains and a C4BP β chain, covalently linked by disulfide bonds at their C-termini (15–17). The assembly of these chains results in a structure, which when resolved by electron microscopy, has been described as spider- or octopus-like (16) (see Figure 1). Each α chain is 70 kDa and composed of 8 internal complement control protein (CCP) domain repeats, while the β chain is 45 kDa and composed of 3 CCP domains. CCP domains are numbered from most distal to the most proximal to the C-terminal disulfide bonds. Four C4BP isoforms have been reported: α7β1, α7β0, α6β1, and α6β0. The 570 kDa multimer α7β1 (7 α chains and 1 β chain) constitutes 80% of the C4BP complexes found in plasma. All β chain-containing C4BP isoforms exist in a high affinity complex with Protein S, a vitamin-K dependent anticoagulant (18–20), which interacts hydrophobically with the β chain CCP1 (21, 22).

Figure 1. Functions of human C4BP.

C4BP, shown here as the major isoform composed of 7 α chains (blue) and 1 β chain (green) linked by disulfide bonds at their C termini, has diverse functions. 1) Canonically, C4BP inhibits the activation of the complement cascade by binding C4b, degrading C4b, and accelerating the decay of the C3 convertase to protect host tissues from uncontrolled complement activation. 2) Microbes hijack C4BP by binding it to their surfaces, where it functions to protect them from complement lysis and clearance by phagocytes, and 3) can modulate complement-independent microbial pathogenesis 4) C4BP promotes cell survival by engaging CD40 and controlled cell death via apoptosis. 5) C4BP controls inflammation in autoimmune diseases and cancer. IAPP = islet amyloid polypeptide; MAC = membrane attack complex; Mo-DC = monocyte-derived dendritic cell; PDAC = pancreatic ductal adenocarcinoma

As an acute-phase reactant, C4BP is transcriptionally upregulated during systemic reaction to infection or tissue injury (23). During the acute-phase response in humans, total plasma concentration of C4BP increases as much as 4-fold, and the C4BP isoforms lacking the β chain increase relative to those containing the β chain (24). The equilibrium between free Protein S (30%) and C4BP-bound Protein S (70%) is important for coagulation pathway homeostasis, and the preferential upregulation of the α7β0 isoform of C4BP during acute-phase conditions helps maintain this balance (24).

Canonically, C4BP inhibits complement activation at the level of the C3 convertase in three ways. First, as its name suggests, C4BP binds to fluid phase and cell surface-bound C4b (25) to prevent formation of the C3 convertase C4bC2b, which in the classical and mannose binding lectin (MBL) pathways requires C4b as a subunit. 4–5 C4b molecules are estimated to bind a single C4BP molecule at once (25). Second, once bound to C4b, C4BP acts as a cofactor for Factor I, which inactivates C4b by cleaving it. Cleavage of C4b generates C4c and C4d, which are functionally inactive, preventing convertase reconstitution. CCPs 1–3 of the C4BP α chain mediate binding to C4b and are required for cofactor activity (17, 26, 27). Third, C4BP accelerates the decay of the C3 convertase by destabilizing and dissociating C2b from the complex (28). Since all of these inhibitory activities are directed at the decay of the C3 convertase C4bC2b, C4BP inhibitory capacity is limited to the classical (28) and MBL (29) pathways in which this convertase is formed.

Exploitation of C4BP for microbial complement resistance

Complement evasion is an important pillar in the coevolution of microbes with humans, as nearly all successful human pathogens have developed strategies to circumvent complement killing (30). One such strategy is capturing and binding human C4BP to a microbial surface ligand to resist complement. Though some microorganisms bind sites on C4BP that overlap with that of C4b, the 7α C4BP multimer remains functionally active to bind and cleave C4b. Over 30 publications (reviewed elsewhere (12, 31); See Table 1) describe C4BP as a mechanism of complement resistance for microbes across taxonomical kingdoms and at a variety of infectious sites. For these microbes, the ability to bind C4BP often correlates with their pathogenic potential (reviewed in (31)). In this section, we highlight the recent developments in pathogens’ complement resistance mediated by C4BP.

Table 1:

C4BP-binding pathogens.

Pathogen		C4BP-binding ligand(s)	Binding domain(s) on C4BP
Bacterial	Bordetella pertussis	FHA(32)	CCP 1–2(32)
	Borrelia afzelii	43 kDa uncharacterized protein(33)	Unknown
	Borrelia burgdorferi
	Borrelia garinii
	Borrelia recurrentis	CihC(34)	Unknown
	Escherichia coli	OmpA(35)	CCP3(35)
	Nontypeable Haemophilus influenzae	Omp P5(36)	CCPs 2, 7(37)
	Leptospira interrogans	LigA, LigB, LcpA(38, 39)	CCPs 7, 8(39)
	Moraxella catarrhalis	UspA1, UspA2(40)	CCPs 2, 5, 7(40)
	Neisseria gonorrhoeae	Porin (Por1A, Por1B)(41) Pili(42)	Porin- CCP1(41) Pili – CCPs 1, 2(42)
	Neisseria meningitidis	PorA(43)	CCPs 2, 3, 6(43)
	Porphyromonas gingivalis	HrgpA(44)	CCPs 1, 6–7(44)
	Prevotella intermedia	Unknown	Unknown
	Salmonella enterica	Rck(45)	CCPs 7, 8(45)
	Staphylococcus aureus	SdrE/Bbp(46)	Unknown
	Streptococcus pneumoniae	LytA(47), PspA(48), PspC(49), PepO(50), Enolase(51)	PspC – CCPs 2,3(49) PepO – CCP8(50) Enolase – CCPs 1,2,8(51) LytA, PspA – unknown
	Streptococcus pyogenes	M proteins (M5(52), M22(53, 54), Protein H(55), M4(56))	Protein H(55), M4(56) - CCPs1, 2
	Yersinia enterocolitica	YadA, Ail(57)	YadA – CCP 1–2(57) Ail – CCP 1–3(57)
	Yersinia pestis	Ail(58)	CCPs 6, 8(58)
	Yersinia pseudotuberculosis	Ail(59)	CCPs 6, 7, 8(59)
Viral	Flaviviruses	NS1(60)	CCPs 2,4,5,8(60)
	Influenza A Virus	HA, NA, M1(61)	CCPs 4–5, 7–8(61)
Eukaryotic	Aspergillus. fumigatus	Enolase(62)	Unknown
	Candida albicans	Pra1(63)	Unknown ligand – CCPs 1, 2(64) Pra1 – CCPs 4, 7, 8(63)
	Loa loa microfilariae	Unknown	Unknown
	Plasmodium falciparum	CSP(65)	CCP 1–2(65)
	Toxoplasma gondii	Unknown	Unknown

Binding of C4BP to the Gram-positive organism Streptococcus pyogenes (group A Streptococcus, GAS) is mediated by a highly variable N-terminal region of the streptococcal M protein family (52). 90% of M protein family members interact with C4BP(66). One member of the M protein family, Protein H, has antiphagocytic properties, which have been attributed to its ability to bind C4BP and the Fc region of human immunoglobulin (Ig-Fc) (54, 55). Binding to complement inhibitors is important for GAS virulence, as C4BP colocalizes with IgG and Protein H in tissues with necrotizing fasciitis caused by GAS (67), and human C4BP transgenic mice exhibit higher GAS burdens and pro-inflammatory cytokine production with enhanced mortality compared to controls without C4BP (68). Recently, a synergy between C4BP and IgG binding to Protein H has been elucidated. When bound to Protein H, Ig-Fc not only inhibits IgG opsonic activity, but also dimerizes Protein H on the bacterial surface, enabling it to bind more molecules of C4BP than when monomeric (67).

Given that several complement proteins are synthesized in the airway epithelium(69), respiratory tract pathogens including Bordetella pertussis and non-typeable Haemophilus influenzae have evolved strategies to evade complement activity. Recently, the OmpA family outer membrane protein, Omp protein 5 (P5) was identified as a H. influenzae ligand for C4BP (36). Along with its polysaccharide capsule, which also confers protection from complement, P5 expression and C4BP binding correlate with H. influenzae resistance to serum (36).

Neisseria gonorrhoeae has one of the most well-studied complement-evasion strategies involving C4BP. N. gonorrhoeae uses its porin and pili to bind human C4BP on its surface. C4BP exhibits cofactor activity in the inactivation of C4b by Factor I (41), markedly inhibiting complement fixation on N. gonorrhoeae. C4BP binding is strongly correlated with N. gonorrhoeae serum resistance, with isolates and mutants that cannot bind C4BP showing sensitivity to serum-mediated lysis (41, 70). The Ram and Blom groups are taking advantage of C4BP binding by N. gonorrhoeae to develop new gonorrhea therapeutics. They engineered a chimeric molecule with C4BP α chain CCPs 1 and 2 fused to the constant portion of IgM, which was multimerized to a hexamer (C4BP-IgM), and found it outcompeted native C4BP binding to the gonococcal surface (71). The C4BP-IgM chimera increases complement activation and subsequent serum bactericidal activity against strains MS11, 1291, 15253, FA1090, and 20 of 26 tested clinical isolates, and it enhances clearance of N. gonorrhoeae from the genital tract of mice that are transgenic for human C4BP (71). C4BP-IgM in conjunction with normal human serum also increased the sensitivity of laboratory strain FA1090 to antibiotics and restored sensitivity to azithromycin for two azithromycin-resistant gonococcal strains, by promoting complement activation, pore formation, and antibiotic entry into the bacterial cell (72). While a similarly generated C4BP-IgG fusion binds gonococci, it does not outcompete native C4BP (71). These studies suggest that C4BP-IgM and antibiotics may be used synergistically to successfully combat drug-resistant gonorrhea.

Flaviviruses (reviewed in (73)) and eukaryotic pathogens also exploit C4BP for complement resistance. The nonstructural protein NS1 from Dengue, West Nile, and yellow fever viruses is displayed on the surface of infected cells and also released into solution. NS1 binds C4BP in solution and recruits it back to the plasma membrane of the infected cell. Binding of C4BP inhibits complement activation on virions and infected cells, allowing evasion of complement control(60). Recent studies show that the opportunistic fungus Aspergillus fumigatus binds C4BP via its enolase, protecting it from complement activation (62, 64). Binding of C4BP for complement evasion also extends to protozoan parasites with primarily intracellular lifestyles that have brief but critical extracellular phases in the blood early in infection. The sporozoite stage of the Plasmodium falciparum parasite resists classical complement activation induced by malarial hyperimmune IgG by binding C4BP via the major surface circumsporozoite protein (CSP) (65). Toxoplasma gondii reduces MAC formation on its surface by binding C4BP and the analogous alternative pathway regulator Factor H(74). The contribution of C4BP to T. gondii survival in vivo, or the ability of C4BP and Factor H to work cooperatively on the surface of the T. gondii, remain open questions (74).

The apparent convergent evolution of diverse C4BP binding ligands and the evolutionary distant pathogens to which they belong (Table 1) underscores the importance of complement resistance via C4BP to microbial pathogenesis.

Complement cascade-independent modulation of microbial pathogenesis

C4BP has recently been implicated in two complement-independent roles related to the interaction of infectious organisms and host cells. Varghese and colleagues reported that C4BP defends against Influenza A virus subtype H1N1 (IAV) without relying on regulation of complement (61). IAV infects through oral or nasal cavities, then hemagglutinin binds to sialic acids in the lung epithelium, where viral particles are endocytosed. C4BP binds to the IAV envelope proteins hemagglutinin, neuraminidase, and matrix protein 1, interactions that mapped to CCP domains 4, 5, 7, and 8. Binding C4BP inhibited the entry of H1N1 pseudotyped particles into lung epithelial cells. Moreover, in line with the concept of C4BP as an anti-inflammatory molecule, C4BP suppressed the pro-inflammatory cytokine storm driven by IAV. IL-12, TNF-α, and NFκB levels were significantly downregulated in C4BP-treated, H1N1-challenged lung epithelial cells. Interestingly, C4BP was also found to bind H3N2 subtype IAV, but promoted viral endocytosis and upregulated proinflammatory cytokine production by lung epithelial cells for this subtype. The authors speculated that the opposing effects of C4BP on H1N1 and H3N2 IAV could be attributed to the structural differences between surface proteins in the two subtypes. Overall, their findings implicate C4BP as an important regulator of IAV replication efficacy by modulating entry into cells, an observation that warrants further study.

Evidence for C4BP functioning independently of complement to benefit a bacterial pathogen was recently uncovered in N. gonorrhoeae. Binding of C4BP to the bacterial surface enhanced its resistance to killing by neutrophils, by limiting neutrophil activation and phagocytosis of N. gonorrhoeae (75). These effects were independent of complement, as shown using serum-free conditions, heat-inactivated serum, and complement component 3 (C3)-depleted serum (75). Curiously, the suppressive activities of C4BP were restricted to N. gonorrhoeae that interacted with neutrophil carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), but not other phagocytic receptors. Given the diverse pathogens that are reported to bind C4BP, many of which encounter phagocytic immune cells during infection (Table 1), it is important to examine if the complement-independent effects of C4BP on N. gonorrhoeae extend to these other organisms. The findings with IAV and N. gonorrhoeae reveal that microbial hijacking of C4BP can affect pathogenesis in ways beyond its long-appreciated role in complement resistance.

Regulation of cell survival and clearance

In serum, C4BP complexes with the vitamin K-dependent glycoprotein and anticoagulant Protein S, which tailors complement deposition and phagocytosis during homeostatic cell clearance to prevent excessive complement activation and inflammation. Protein S binds to negatively charged phospholipids on apoptotic cells including neutrophils (76–78). During early apoptosis, the binding of free Protein S stimulates phagocytosis of apoptotic cells by macrophages (79), which is important for apoptotic cell clearance. During mid to late apoptosis, complement activation is initiated on the apoptotic cell surface, and the C4BP-Protein S complex binds (80). In contrast to Protein S alone, C4BP-Protein S does not promote phagocytosis (81), and instead is thought to benefit the host by replacing the membrane-bound regulators lost during apoptosis, and by limiting C3 and C9 deposition to avoid complement activation, prevent necrosis, and promote controlled cell death via apoptosis (80).

C4BP influences cell survival in a complement-independent manner by interacting with CD40, a receptor found on diverse cell types including antigen presenting cells and epithelial cells. In tonsillar tissue, CD40 on B cells directly binds the α-chain of C4BP in a manner that mimics signaling from CD40 ligand (CD40L) (82). C4BP induces B cell proliferation, upregulates CD54 and CD86 expression, with IL-4 stimulation induces isotype switching to IgE, and promotes signaling through NFκB and p38 MAP kinase (82). However, C4BP binds to a distinct site on CD40 and does not compete with CD40L. In germinal centers where CD40L is not detectable, C4BP may phenocopy CD40 ligation to promote B cell survival. C4BP similarly modulates epithelial cell survival in the bile duct. Here, C4BP complexes with soluble CD40L, preventing it from ligating to CD40, which abrogates apoptosis of cholangiocytes and permits cell survival (83). Since increased apoptosis of cholangiocytes is implicated in diseases such as primary biliary cirrhosis, C4BP is an important down-regulator of cholangiocyte apoptosis, and critical for biliary duct integrity. Thus, C4BP plays a significant role in the regulation of cell survival, in a complement-independent manner.

Modulation of inflammation in cancer and chronic disease

C4BP is a regulator of excessive inflammation in chronic disease, which protects healthy host cells. However, C4BP can also protect tumor cells from host immune cell clearance. In this way, C4BP has both beneficial and detrimental functions relating to inflammation in chronic disease.

The α chain of C4BP exhibits complement-independent antitumor immunity in the pancreas, where its expression is correlated with tumor regression and more favorable outcomes for pancreatic ductal adenocarcinoma (PDAC). In vivo mouse models have shown that, similarly to B cells in the tonsil, the α7β0 form of C4BP (hereafter called C4BPα) binds to CD40 on B cells and other antigen presenting cells in the pancreas. This promotes accumulation of antitumor T cells at the periphery of PDACs (84). In another antitumor capacity, C4BPα is expressed intracellularly in colorectal cancer cells. Expression of C4BPα with certain mutations drives NFκB-dependent apoptosis in the tumor cells, and correlates with improved patient survival outcomes (85).

However, C4BP can also be tumor-promoting when functioning as a complement inhibitor. C4BP protects ovarian adenocarcinoma cells from complement activation by binding the surface via CCP4, and retaining functional cofactor activity for Factor I- mediated inactivation of C4b (86). In another tumor-promoting function, C4BPα expression is induced by the oncogenic Hepatitis B virus. C4BPα binds to the surface of hepatocellular carcinoma cells, thereby protecting the cells from complement-dependent cytotoxicity and promoting hepatoma survival (87).

C4BP also exhibits anti-inflammatory activity in the context of autoimmunity. Extracellular DNA elicits autoantibodies and complement and is implicated in autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus (SLE). C4BP binds DNA via a positively charged patch of amino acids in α chain CCP2, capturing free DNA at the necrotic cells surface, and thereby limiting the inflammatory potential of necrosis (88).

C4BP helps limit the development of autoimmunity in SLE, a disease in which uncontrolled inflammation leads to tissue damage, often in the kidney (lupus nephritis). Underscoring the importance of C4BP in SLE, C4BP lacking the β chain (C4BP (β−)) protects lupus-prone mice from nephritis by downregulating immunopathogenic cell infiltration into the kidney (89). Moreover, individuals with active lupus flares have lower levels of C4BP in plasma (90, 91). Additionally, the CCP6 domain of C4BP(β−) reprograms monocyte-derived dendritic cells (Mo-DCs) isolated from lupus nephritis patients from a pro-inflammatory to an anti-inflammatory phenotype, as shown by downregulation of surface activation markers and pro-inflammatory cytokines TNF-α and IL-12 (92, 93). Interestingly, the β chain interferes with this function, but a multimer made of solely CCP6 and the oligomerization domains of C4BP is sufficient to recapitulate the function of limiting of lupus nephritis in animal models (89, 93). Since β chain-deficient forms of C4BP are upregulated during the acute-phase, this may represent a mechanism by which C4BP protects the kidney in the context of SLE.

While most C4BP is produced in the liver, C4BP is also secreted from the islet cells of the pancreas, where it has a cytoprotective effect. Here, C4BP binds to islet amyloid polypeptide (IAPP) (11), a protein that is co-secreted with insulin. For individuals with type 2 diabetes, IAPP leads to the formation of amyloid deposits, which induce inflammasome activation of beta cells (94). C4BP localizes to these deposits and neutralizes the activity of IAPP, which blocks fibrillation of IAPP and prevents IAPP-mediated IL-1β production and IAPP-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation, protecting beta cell function and viability (94). Recently, evidence of C4BP as an inhibitor of NLRP3 inflammasome activation has been extended beyond IAPP. C4BP binds to and co-internalizes into human primary macrophages with monosodium urate crystals and silica (drivers of inflammation in gout and silicosis, respectively), where it prevents NLRP3 inflammasome activation by protecting against lysosomal damage (95). In these ways, C4BP is a critical modulator of inflammation, whether to host benefit or detriment, in chronic conditions of diverse etiology.

Conclusions

C4BP is emerging as a broad-acting molecule with diverse functions (see Figure 1). Its contribution to controlling inflammation can have beneficial or deleterious effects for human health, as C4BP protects healthy host cells, tumor cells, and pathogens alike. In an extension of the well-known ability of C4BP to protect microorganisms from complement-mediated killing, recent reports characterize C4BP as a complement-independent modulator of virulence for pathogenic microorganisms.

Open questions remain about how C4BP may modulate the efficacy of immunotherapies that rely on complement activation to kill malignant cells or pathogens, such as rituximab for B cell malignancies or vaccines for pathogens (96). There is promising evidence that C4BP may be harnessed in modified forms to be used therapeutically, such as C4BP-IgM for treatment of N. gonorrhoeae (71, 72) or M. catarrhalis (97), or as a CCP6 multimer for the treatment of SLE (93). Furthermore, there may be unappreciated complement-independent effects of C4BP for other microbes. For example, C4BP reduces invasion of both H1N1 IAV and N. gonorrhoeae, raising the question of whether C4BP may broadly inhibit interactions of pathogens with host cells. The contributions of C4BP to homeostasis and chronic conditions of infectious and non-infectious etiologies will continue to be uncovered, revealing new perspectives on the balance between complement-dependent and complement-independent activities of C4BP.