The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and β-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the β-arrestin pathway. Association of C5L2 with β-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the β-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.     

Structure-function relationships of human C5a and C5aR

Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four alpha helices of C5a, we have defined the ability of these peptides to reduce binding of (125)I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H(2)O(2) production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a.     

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or β-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a–C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.     

The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.     

Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.     

Receptors for complement C5a. The importance of C5aR and the enigmatic role of C5L2

Complement component C5a is one of the most potent inflammatory chemoattractants and has been implicated in the pathogenesis of numerous inflammatory diseases. C5a binds two receptors, C5aR and C5L2. Most of the C5a functional effects occur through C5aR, and the pharmaceutical industry has focused on this receptor for the development of new anti-inflammatory therapies. We used a novel approach to generate and test therapeutics that target C5aR. We created human C5aR knock-in mice, and used neutrophils from these to immunize wild-type mice. This yielded high-affinity blocking mAbs to human C5aR. We tested these anti-human C5aR mAbs in mouse models of inflammation, using the human C5aR knock-in mice. These antibodies completely prevented disease onset and were also able to reverse established disease in the K/B x N arthritis model. The physiological role of the other C5a receptor, C5L2 is still unclear, and our studies with blocking mAbs to human C5L2 have failed to demonstrate a clear functional role in signaling to C5a. The development of effective mAbs to human C5aR is an alternative approach to drug development, for this highly attractive target.     

Inhibition of C5-cytosine-DNA-methyltransferases

Changes in the methylation pattern of genomic DNA, particularly hypermethylation of tumor suppressor genes, occur at early stages of tumor development. Errors in DNA methylation contribute to both initiation and progression of various cancers. This stimulates significant interest in searching for inhibitors of C5-DNA-methyltransferases (MTases). Here we review the known nucleoside mechanism-based reversible and irreversible inhibitors of the MTases, as well as non-nucleoside ones, and discuss their inhibitory mechanisms and application for MTase investigations and cancer therapy.     

Preservation of C3, C5, and C5a functional activities by a new radiolabeling method; demonstration of C5 products in complement-activated serum.

A new method is described for the radiolabeling of C3, C5, and C5a. Using a methylation procedure we have labeled C3, C5, and C5a with 14CH20, achieving high specific activity, without loss of hemolytic activity in the case of C3 and C5, and in the case of C5a, full retention of chemotactic and enzyme releasing activities. The distribution of radiolabel in the alpha and beta chains of C5 was 75 and 25%, respectively. Using intact labeled C5 the fragmentation pattern in zymosan-activated human serum, assessed by gel filtration and electrophoresis, is complex, yielding multiple fragmentation products. The labeling method described in this paper yield materials that have many advantages over the currently used labeling procedures.     

Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor.

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C-terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N-terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site-directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (beta-glucuronidase release). Mutants that contained the single residue substitutions Ile-6-->Gly or Tyr-13-->Gly were reduced in potency to 4-30% compared with wild-type rC5a. Other single-site glycine substitutions at positions Leu-2, Ala-10, Lys-4, Lys-5, Glu-7, Glu-8, and Lys-14 showed little effect on C5a potency. The double mutant, Ile-6-->Gly/Tyr-13-->Gly, was reduced in potency to < 0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in alpha-helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N-terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.     

A Pro-Inflammatory Role of C5L2 in C5a-Primed Neutrophils for ANCA-Induced Activation

Background

The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a and its receptor on neutrophils, CD88, play a central role. The functional role of the second receptor of C5a, C5L2, remains unclear. In the current study, we investigated the role of C5L2 in C5a-primed neutrophils for ANCA-induced activation.

Methods

The effect of blocking C5L2 by anti-human C5L2 blocking antibody were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on membrane-bound proteinase 3 (mPR3) and concentration of myeloperoxidase (MPO) in supernatant of C5a-primed neutrophils. An antagonist for CD88 was also employed.

Results

Blocking C5L2 resulted in a significantly decreased MPO concentration in the supernatant of C5a-primed neutrophils. mPR3 expression increased from 209.0±43.0 in untreated cells to 444.3±60.8 after C5a treatment (P<0.001), and decreased to 375.8±65.44, 342.2±54.3 and 313.7±43.6 by pre-incubating blocking C5L2 antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-priming group, P<0.001, P<0.001, and P<0.001), respectively. In C5a-primed neutrophils, subsequently activating with MPO-ANCA-positive IgG, the MFI value was 425.8±160.6, which decreased to 292.8±141.2, 289.7±130.0 and 280.3±136.4 upon pre-incubation with mouse anti-human C5L2 blocking antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-primed neutrophils, for MPO-ANCA-positive IgG-induced activation, P<0.05, P<0.05, and P<0.05), respectively. Blocking C5L2 also resulted in significantly decreased C5a-primed neutrophils for PR3-ANCA-positive IgG-induced activation. Moreover, the lactoferrin concentration in the supernant significantly decreased in pre-incubation with anti-human C5L2 blocking antibody, compared with C5a-primed neutrophils induced by PR3- or MPO-ANCA-positive IgG.

Conclusions

C5L2 may be implicated in the pro-inflammatory role in C5a-primed neutrophils for ANCA-induced activation.     

Complement component 5a (C5a)

The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.     

Structure and characterization of a high affinity C5a monoclonal antibody that blocks binding to C5aR1 and C5aR2 receptors

C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a–C5aR1 receptor are well defined, whereas C5a–C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement–mediated bacterial cell killing. Unlike other anti–C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a–C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia–reperfusion injury.     

Mapping the ligand-binding site on the C5a receptor: arginine74 of C5a contacts aspartate282 of the C5a receptor.

The interaction between the anaphylatoxin C5a and its receptor involves two distinct sites. One site is formed by acidic residues at the receptor N-terminus and contributes to only ligand binding. The second site, responsible for activation, is less well defined. In this study, we demonstrate that the receptor residue D(282), near the extracellular face of transmembrane domain VII, is a component of the second ligand-binding site. Mutation of D(282) to A decreases the sensitivity of the receptor to activation by intact C5a but not by its less potent metabolite, C5adR(74), which lacks the C-terminal arginine(74). The mutation of the R(74) residue of C5a to A causes a 60-fold decrease in wild-type receptor sensitivity, but only a 2-fold decrease for the receptor mutated at D(282). In contrast, the mutation of R(74) to D makes C5a completely inactive on both wild-type and A(282) C5a receptors. The mutation of D(282) to R partly restores the response to C5a[D(74)], which is a more effective ligand than C5a at the mutant receptor. A peptide mimic of the C5a activation domain with a C-terminal R potently activates the wild type but is only a weak agonist at the mutant D(282)R-C5a receptor. Conversely, a peptide with D at the C-terminus is a more effective activator of D(282)R than wild-type C5a receptors. These data indicate that the R(74) side chain of C5a makes an interaction with receptor D(282) that is responsible for the higher potency of intact C5a versus that of C5adR(74).     

Modeling molecular mechanisms of binding of the anaphylatoxin C5a to the C5a receptor

This study presents the 3D model of the complex between the anaphylatoxin C5a and its specific receptor, C5aR. This is the first 3D model of a G-protein-coupled receptor (GPCR) complex with a peptide ligand deduced by a molecular modeling procedure analyzing various conformational possibilities of the extracellular loops and the N-terminal segment of the GPCR. The modeling results indicated two very different ways of interacting between C5a and C5aR at the two interaction sites suggested earlier based on the data of site-directed mutagenesis. Specifically, C5a and C5aR can be involved in "mutual-induced fit", where the interface between the molecules is determined by both the receptor and the ligand. The rigid core of the C5a ligand selects the proper conformations of the highly flexible N-terminal segment of C5aR (the first interaction site). At the same time, the binding conformation of the flexible C-terminal fragment of C5a is selected by well-defined interactions with the TM region of the C5aR receptor (the second interaction site). The proposed 3D model of C5a/C5aR complex was built without direct use of structural constraints derived from site-directed mutagenesis reserving those data for validation of the model. The available data of site-directed mutagenesis of C5a and C5aR were successfully rationalized with the help of the model. Also, the modeling results predicted that the full-length C5a and C5a-des74 metabolite would have different binding modes with C5aR. Modeling approaches employed in this study are readily applicable for studies of molecular mechanisms of binding of other polypeptide ligands to their specific GPCRs.     

Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts

C5a and C5a des Arg are chemotactic factors for inflammatory cells but it is not known whether these agents are chemoattractants for fibroblasts. Accordingly, C5a, purified from zymosan-activated human, and C5a des Arg, prepared by incubating C5a with immobilized porcine carboxypeptidase B, were studied for fibroblast chemotactic activity. We observed that both C5a and C5a des Arg stimulated human skin fibroblasts and fetal bovine ligament fibroblasts to migrate in a concentration-dependent fashion, and that the migratory responses were similar in magnitude to the responses achieved with optimal concentrations of two known fibroblast chemoattractants, platelet-derived growth factor and human fibrinopeptide B. The peak responses to C5a and C5a des Arg occurred at approximately 10(-9)M. With ligament fibroblasts, there was a greater response to C5a des Arg than to C5a, but with human fibroblasts there was no difference. Cochemotaxin, which enhances the chemotactic activity of C5a des Arg for neutrophils, had no effect on C5a des Arg fibroblast chemotactic activity but appeared to increase the fibroblast chemotactic activity of C5a. These results indicate that the effects of C5a and C5a des Arg in vivo may extend to the recruitment of mesenchymal cells. Moreover, the findings represent another example of an activity retained by C5a after removal of its carboxyl terminal arginine.     

C5L2 is required for C5a-triggered receptor internalization and ERK signaling

C5L2 is a receptor that binds to C5a and belongs to the family of G protein-coupled receptors, but its role in physiological C5a-mediated responses remains under debate. Here we show that, like the canonical C5a receptor C5aR, C5L2 plays a pro-inflammatory role in a murine model of acute experimental colitis. We demonstrate that C5L2 physically interacts with C5aR and is required for optimal C5a-mediated C5aR internalization and associated ERK activation. Abrogation of C5a-induced receptor internalization by treatment with the dynamin inhibitor dynasore^TM impaired C5a-induced MEK and ERK signaling. Although the presence of C5aR alone was sufficient to recruit the scaffold protein β-arrestin1 to the cell membrane in response to C5a stimulation, it was inadequate to mediate AP2 recruitment and subsequent C5aR internalization. Expression of C5L2 allowed normal internalization of C5aR in response to C5a stimulation, followed by normal ERK signaling. Thus, our work reveals an essential role for C5L2 in C5a-triggered, AP2-dependent C5aR internalization and downstream ERK signaling.     

Microvascular effects of anaphylatoxins C3a and C5a

The direct microvascular effects of human C3a and C5a were investigated by using hamster cheek pouches prepared for intravital microscopy. Topical application of 10 nM C3a resulted in pronounced microcirculatory alterations, characterized by vasoconstriction, platelet aggregation, and an increase in macromolecular leakage at postcapillary venules, as assessed by extravasation of intravascular fluorescein-labeled dextran (m.w. 150,000). Exposure of the preparation to 500 nM COOH-terminal octapeptide analogs of C3a resulted in a microvascular response almost identical to that of C3a, supporting the view that the active site of this anaphylatoxin resided within the COOH-terminal portion. Changes similar to those caused by C3a were also induced by 20 or 100 nM C5a and, in addition, the higher concentration of C5a caused accumulation of polymorphonuclear leukocytes (PMNL) in small venules and somewhat prolonged vascular leakage from venules exhibiting PMNL accumulation. Histamine was found to partially mediate the vascular leakage induced by C3a and the initial (first 5 min) permeability response to the high concentration of C5a, whereas the subsequent leakage induced by the latter anaphylatoxin was unaffected by mepyramine pretreatment. In neutropenic and mepyramine-treated animals exposed to the high concentration of C5a, a partial reduction of both the early and the subsequent vascular leakage was seen, indicating that accumulated PMNL play a role in the prolonged phase of leakage. The pronounced microvascular alterations induced by low concentrations of C3a and C5a strengthen the view that these anaphylatoxins act as mediators of inflammatory reactions in which the complement system is activated.