cDNA Cloning and Characterization of Rat C5a Anaphylatoxin Receptor

Activation of the complement cascade plays an esssential role in the early stages of inflammation. C5a and its receptor are particularly active in anaphylaxis. To determine the pathological roles played by C5a and C5a receptor (C5aR) in rats, we cloned C5aR cDNA and analyzed distribution of its mRNA in various organs including lung from an LPS-stimulated rat. Furthermore, we generated a polyclonal antiserum which specifically recognizes rat C5aR, as confirmed by its specific interaction with cells transfected with rat C5aR cDNA.     

Characterization of a polymorphism in the coding region of the human C5a anaphylatoxin receptor

 A polymorphism was identified in the coding region of the human C5a anaphylatoxin receptor gene leading to C to T transition at nucleotide position 450 (a silent substitution in the Ala¹⁵⁰ codon, GCC to GCT). Its distribution was studied in a population of healthy volunteers from the Québec city region (prevalence of 2.8%) and among patients with end-stage renal failure who had previously undergone renal graft (prevalence 1.4%, not significantly different from that of the control group). This new marker provides a valuable tool to assess the risk for putative C5a-associated disorders with genetic determinism. Received: 20 November 1998 / Revised: 24 February 1999     

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.     

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.     

Design and optimization of aniline-substituted tetrahydroquinoline C5a receptor antagonists

A series of aniline-substituted tetrahydroquinoline C5a receptor antagonists were discovered. A functionality requirement of ortho substitution on the aniline was revealed. Secondary anilines, in general, outperformed tertiary analogs in inhibition of C5a-induced calcium mobilization. Further enhancement of activity was realized in the presence of an ortho hydroxyalkyl side chain. The functional IC₅₀ of selected analogs was optimized to the single-digit nanomolar level.     

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.     

Different endocytosis pathways of the C5a receptor and the N-formyl peptide receptor

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N-formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re-phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand-dependent redistribution of C5aR-EGFP from the plasma membrane to LAMP2-positive compartments, whereas FPR-EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down-regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR-mediated chemotaxis was not observed.     

Primary structure and functional characterization of rat C5a: an anaphylatoxin with unusually high potency.

The anaphylatoxin C5a is a pro-inflammatory factor generated from C5 during complement activation. C5a derived from rat C5 exhibits significantly greater potency compared to C5a from other species. Rat C5a was 25-fold more potent than human C5a for eliciting spasmogenic contraction of guinea pig ileum. Proteolytic removal of the C-terminal arginine of C5a (C5adesArg) reduced spasmogenic potency of rat C5a by only 4-fold compared to a 3,000-fold reduction for human C5adesArg. In addition, rat C5adesArg was 50-fold more potent than human C5adesArg in a guinea pig vascular permeability (in vivo) assay and as a chemotactic factor for human neutrophils. C5a and C5adesArg were purified from zymosan-activated rat serum. Rat C5a, like human C5a, is glycosylated but contains 77 amino acid residues instead of the 74 residues of human C5a. Comparison of the primary structures of rat and human C5a indicated differences at 30 positions including an insert of 3 residues (LLH) in the rat molecule between residue positions 3 and 4 in human C5a. Insertion of residues LLH between Gln-3 and Lys-4 in a recombinant human C5a molecule using site-directed mutagenesis failed to enhance potency. Synthetic C-terminal analogues of rat C5a proved to be measurably more potent than the corresponding human C5a analogues (Ember JA et al., 1993, Protein Sci 2(Suppl 1):159 [Abstr]). We conclude that multiple sequence differences in the C-terminal effector portion and/or elsewhere in rat C5a, but not the LLH insert, account for the significant enhancement in potency of rat C5a over C5a from other species.     

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.     

C5aR-mediated myocardial ischemia/reperfusion injury

The complement system activation can mediate myocardial ischemia and reperfusion (I/R). Inhibition of C5a activity reveals attenuation of I/R-induced myocardial infarct size. However, the contribution of C5a receptor (C5aR) to I/R injury remains to be unknown. Here, we reported that both mRNA and protein for the C5aR were constitutively expressed on cardiomyocytes and upregulated as a function of time after I/R-induced myocardial cell injury in mice. Blockade of C5aR markedly decreased microvascular permeability in ischemic myocardial area and leukocyte adherence to coronary artery endothelium. Importantly, the blocking of C5aR with an anti-C5aR antibody was associated with inhibition in activation of protein kinase C delta (PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. These data provide evidence that C5aR-mediated myocardial cell injury is an important pathogenic factor, and that C5aR blockade may be useful therapeutic targets for the prevention of myocardial I/R injury.     

Loss of C3a and C5a receptors promotes adipocyte browning and attenuates diet-induced obesity via activating inosine/A2aR pathway

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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.     

Complement anaphylatoxin C5a neuroprotects through mitogen-activated protein kinase-dependent inhibition of caspase 3

We previously reported that pretreatment of murine cortico-hippocampal neuronal cultures with the complement-derived anaphylatoxin C5a, protects against glutamate neurotoxicity. In this study we explored the potential mechanisms involved in C5a-mediated neuroprotection. We found that C5a neuroprotects in vitro through inhibition of apoptotic death because pretreatment with human recombinant (hr)C5a prevented nuclear DNA fragmentation coincidental to inhibition of the pro-apoptotic caspase 3 activity mediated by glutamate treatment. Also, hrC5a-mediated responses appeared to be receptor-mediated because pretreatment of cultures with the specific C5a receptor antagonist C177, prevented hrC5a-mediated neuroprotection. Based on this evidence, we further explored possible signaling pathways involved in hrC5a inhibition of caspase 3 activation and apoptotic neuronal death. We found that treatment of cultures with the mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059 prevented hrC5a-mediated inhibition of caspase 3 and apoptotic neuron death. MAPK pathways, whose activation by hrC5a is inhibited by PD98059 and C177, include the extracellular signal-regulated kinase (ERK)2 and, to a lesser extent, ERK1. The study suggests that C5a may protect against glutamate-induced apoptosis in neurons through MAPK-mediated regulation of caspase cascades.     

Simplified modeling approach suggests structural mechanisms for constitutive activation of the C5a receptor

Molecular modeling of conformational changes occurring in the transmembrane region of the complement factor 5a receptor (C5aR) during receptor activation was performed by comparing two constitutively active mutants (CAMs) of C5aR, NQ (I124N/L127Q), and F251A, to those of the wild-type C5aR and NQ-N296A (I124N/L127Q/N296A), which have the wild-type phenotype. Modeling involved comprehensive sampling of various rotations of TM helices aligned to the crystal template of the dark-adapted rhodopsin along their long axes. By assuming that the relative energies of the spontaneously activated states of CAMs should be lower or at least comparable to energies characteristic for the ground states, we selected the plausible models for the conformational states associated with constitutive activation in C5aR. The modeling revealed that the hydrogen bonds between the side chains of D82-N119, S85-N119, and S131-C221 characteristic for the ground state were replaced by the hydrogen bonds D82-N296, N296-Y300, and S131-R134, respectively, in the activated states. Also, conformational transitions that occurred upon activation were hindered by contacts between the side chains of L127 and F251. The results rationalize the available data of mutagenesis in C5aR and offer the first specific molecular mechanism for the loss of constitutive activity in NQ-N296A. Our results also contributed to understanding the general structural mechanisms of activation in G-protein-coupled receptors lacking the "ionic lock", R(3.50) and E/D(6.30). Importantly, these results were obtained by modeling approaches that deliberately simplify many elements in order to explore potential conformations of GPCRs involving large-scale molecular movements.     

Solution structure of a unique C5a semi-synthetic antagonist: implications in receptor binding.

The tertiary structure of a unique C5a receptor antagonist was determined by two-dimensional NMR spectroscopy. The core domain of this 8-kDa antagonist exists as an antiparallel helical bundle, similar to recombinant human (rh)-C5a. However, unlike C5a, the antagonist's C terminus was found to be conformationally restricted along a groove between helices one and four in the core domain. This conformational restriction situates C-terminal D-Arg 75 in a wedge between core residues Arg 46 and His 15. Correlation of the antagonist's tertiary structure with point mutation analysis revealed the formation of a positively charged contiguous contact surface comprised of D-Arg 75, Arg 46, Lys 49, and His 15. The significance of this surface in generating antagonist properties implies a single binding site with the C5a receptor and provides a structural template for drug design.     

Structural models for the complex of chemotaxis inhibitory protein of Staphylococcus aureus with the C5a receptor

The study presents structural models for the complex of the chemotaxis inhibitory protein of Staphylococcus aureus, CHIPS, and receptor for anaphylotoxin C5a, C5aR. The models are based on the recently found NMR structure of the complex between CHIPS fragment 31-121 and C5aR fragment 7-28, as well as on previous results of molecular modeling of C5aR. Simple and straightforward modeling procedure selected low-energy conformations of the C5aR fragment 8-41 that simultaneously fit the NMR structure of the C5aR 10-18 fragment and properly orient the NMR structure of CHIPS_31-121 relative to C5aR. Extensive repacking of the side chains of CHIPS_31-121 and C5aR_8-41 predicted specific residue-residue interactions on the interface between CHIPS and C5aR. Many of these interactions were rationalized with experimental data obtained by site-directed mutagenesis of CHIPS and C5aR. The models correctly showed that CHIPS binds only to the first binding site of C5a to C5aR not competing with C5a fragment 59-74, which binds the second binding site of C5aR. The models also predict that two elements of CHIPS, fragments 48-58 and 97-111, may be used as structural templates for potential inhibitors of C5a.     

RP S19 C-terminal peptide trimer acts as a C5a receptor antagonist

We have demonstrated that ribosomal protein S19 (RP S19) polymer, when crosslinked between Lys122 and Gln137 by activated coagulation factor XIII, acts as a C5a receptor (C5aR) antagonist/agonist. Based on experimental data obtained using RP S19 analog peptide and recombinant protein monomer, we suggested that L₁₃₁DR, I₁₃₄AGQVAAAN and K₁₄₃KH moieties in the RP S19 C‐terminus act in, respectively, C5aR binding, penetration of the plasma membrane, and interaction with either an apoptosis-inducing molecule in neutrophils (delta lactoferrin) or a calcium channel-activating molecule (annexin A3) to induce the p38 MAPK pathway in macrophages. Recently, we observed RP S19 trimer in serum. To study the effects of this RP S19 trimer on C5aR, we prepared mutant RP S19 C‐terminal peptide (RP S19^122-145) dimer and trimer, and examined their chemotactic activities and signal transduction pathways in human C5aR-overexpressing squamous cell carcinoma HSC-1 (HSC-1^C5aR) cells using 24 trans-well chamber and western blotting assays, respectively. HSC-1^C5aR cells were attracted by RP S19^122-145 dimer and vice versa by RP S19^122-145 trimer. The RP S19^122-145 dimer-induced attraction was competitively blocked by pre-treatment with RP S19^122-145 trimer. Moreover, RP S19^122-145 trimer-induced p38 MAPK phosphorylation was stronger than RP S19^122-145 dimer-induced p38 MAPK phosphorylation. RP S19^122-145 trimer appeared to act as a C5aR antagonist. The agonistic and antagonistic effects of RP S19^122-145 dimers and trimers were reflected by monocytic, THP-1-derived macrophage-like cells. Unlike the C5aR agonist C5a, which acts at the inflammation phase of acute inflammation, RP S19 trimer might act as a C5aR antagonist at the resolution phase.     

Allosterism in human complement component 5a (hC5a): a damper of C5a receptor (C5aR) signaling

The phenomena of allosterism continues to advance the field of drug discovery, by illuminating gainful insights for many key processes, related to the structure–function relationships in proteins and enzymes, including the transmembrane G-protein coupled receptors (GPCRs), both in normal as well as in the disease states. However, allosterism is completely unexplored in the native protein ligands, especially when a small covalent change significantly modulates the pharmacology of the protein ligands toward the signaling axes of the GPCRs. One such example is the human C5a (^hC5a), the potent cationic anaphylatoxin that engages C5aR and C5L2 to elicit numerous immunological and non-immunological responses in humans. From the recently available structure–function data, it is clear that unlike the mouse C5a (^mC5a), the ^hC5a displays conformational heterogeneity. However, the molecular basis of such conformational heterogeneity, otherwise allosterism in ^hC5a and its precise contribution toward the overall C5aR signaling is not known. This study attempts to decipher the functional role of allosterism in ^hC5a, by exploring the inherent conformational dynamics in ^mC5a, ^hC5a and in its point mutants, including the proteolytic mutant des-Arg⁷⁴-^hC5a. Prima facie, the comparative molecular dynamics study, over total 500 ns, identifies Arg⁷⁴-Tyr²³ and Arg³⁷-Phe⁵¹ “cation-π” pairs as the molecular “allosteric switches” on ^hC5a that potentially functions as a damper of C5aR signaling.