CiC3-1a-mediated chemotaxis in the deuterostome invertebrate Ciona intestinalis (Urochordata)

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a(59-76)) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a(59-75)) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is G(i) protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3'-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.     

Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species

C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.     

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.     

Sulfation of tyrosine 174 in the human C3a receptor is essential for binding of C3a anaphylatoxin

The complement anaphylatoxin C3a and its cellular seven-transmembrane segment receptor, C3aR, are implicated in a variety of pathological inflammatory processes. C3aR is a G-protein-coupled receptor with an exceptionally large second extracellular loop of 172 amino acids. Previously reported deletion studies have shown that at least part of this region plays a critical role in binding C3a. Our data now demonstrate that five tyrosines in the second extracellular loop of the C3aR are posttranslationally modified by the addition of sulfate. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 184, 188, 317, and/or 318 does not affect ligand binding or signal transduction. However, when tyrosine 174 is mutated to phenylalanine, binding of native C3a is completely blocked. This variant efficiently mobilizes calcium in response to synthetic C3a agonist peptides, but not to native C3a. This finding is consistent with a two-site model of ligand association typical of many peptide ligand-receptor interactions and identifies sulfotyrosine 174 as the critical C3a docking site. Tyrosine sulfation in the amino-terminal extracellular domain has been shown to be important in several other seven-transmembrane segment receptors. Our data now demonstrate that tyrosine sulfate in other extracellular domains can function for ligand interactions as well.     

Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis

The recent identification of complement components in deuterostome invertebrates has indicated the presence of a complement system operating via an alternative pathway in echinoderms and tunicates and via a MBL-mediated pathway thus far identified only in tunicates. Here, we report the isolation of two C3-like genes, CiC3-1 and CiC3-2, from blood cell total RNA of the ascidian Ciona intestinalis. The deduced amino acid sequences of both Ciona C3-like proteins exhibit a canonical processing site for alpha and beta chains, a thioester site with an associated catalytic histidine and a convertase cleavage site, thus showing an overall similarity to the other C3 molecules already characterized. Southern blotting analysis indicated that each gene is present as a single copy per haploid genome. In situ hybridization experiments showed that both CiC3-1 and CiC3-2 are expressed in one type of blood cell, the compartment cells. Two polyclonal antibodies, raised against two deduced peptide sequences in the alpha chain of CiC3-1 and CiC3-2, allowed the identification by Western blot of a single band in the blood serum, of about M(r)150,000. A phylogenetic tree, based on the alignment of CiC3-1 and CiC3-2 with molecules of the alpha(2)-macroglobulin superfamily, indicated that the Ciona C3s form a cluster with Halocynthia roretzi C3. The phylogenetic analysis also suggested that the duplication event from which the CiC3-1 and CiC3-2 genes originated occurred in the urochordate lineage after the separation of the Halocynthia and Ciona ancestor.     

Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function

The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.     

Chimeric receptors of the human C3a receptor and C5a receptor (CD88)

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.     

Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.     

Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.     

Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.     

Residues 10-18 within the C5a receptor N terminus compose a binding domain for chemotaxis inhibitory protein of Staphylococcus aureus

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by the majority of S. aureus strains and is a potent inhibitor of C5a- and formylated peptide-mediated chemotaxis of neutrophils and monocytes. Recently, we reported that CHIPS binds to the C5a receptor (C5aR) and the formylated peptide receptor, thereby blocking activation by C5a and formylated peptides, respectively. The anaphylatoxin C5a plays an important role in host immunity and pathological inflammatory processes. For C5a a two-site binding model is proposed in which C5a initially binds the C5aR N terminus, followed by interaction of the C5a C-terminal tail with an effector domain on the receptor. We have shown here that CHIPS does not affect activation of the C5aR by a peptide mimic of the C5a C terminus. Moreover, CHIPS was found to bind human embryonic kidney 293 cells expressing only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal expression system revealed that the binding site of CHIPS is contained in a short stretch of 9 amino acids (amino acids 10-18), of which the aspartic acid residues at positions 10, 15, and 18 plus the glycine at position 12 are crucial. Binding studies with C5aR/C3aR and C5aR/IL8RA chimeras confirmed that CHIPS binds only to the C5aR N terminus without involvement of its extracellular loops. CHIPS may provide new strategies to block the C5aR, which may lead to the development of new C5aR antagonists.     

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.     

Activated human T lymphocytes express a functional C3a receptor

The C3a molecule is an anaphylatoxin of the C system with a wide spectrum of proinflammatory effects predominantly on cells of myeloid origin. In this study we investigated the expression of the high affinity receptor for C3a (C3aR) in human T lymphocytes using receptor-specific mAb. C3aR expression was detected in CD4(+) and CD8(+) blood- or skin-derived T cell clones (TCC) from birch pollen-sensitized patients with atopic dermatitis. No significant difference in C3aR expression in CD4(+) or CD8(+) TCCs could be observed. In contrast to C3a(desArg), C3a led to a transient calcium flux in TCCs expressing the C3aR, whereas C3aR-negative TCCs were unreactive. Circulating T cells from patients suffering from severe inflammatory skin diseases expressed the C3aR, whereas no expression of C3aR could be found in unstimulated T lymphocytes from patients with mild inflammatory skin diseases or from healthy individuals. Type I IFNs, which are potent stimulators of cellular immunity, were identified as up-regulators of C3aR expression in vitro in freshly isolated or cloned T lymphocytes. Moreover, C3aR(+) T cells were found at the sites of injection in IFN-beta-treated patients with multiple sclerosis. These data provide direct evidence for the expression of C3aR on activated human T lymphocytes; this may point to a biological function of C3a in T cell-dependent diseases.     

Antisense knockdown of sphingosine kinase 1 in human macrophages inhibits C5a receptor-dependent signal transduction, Ca2+ signals, enzyme release, cytokine production, and chemotaxis

The anaphylatoxin C5a is produced following the activation of the complement system and is associated with a variety of pathologies, including septic shock and adult respiratory distress syndrome, and with immune complex-dependent diseases such as rheumatoid arthritis. C5a has been shown to regulate inflammatory functions by interacting with its receptor, C5aR, which belong to the rhodopsin family of seven-transmembrane GPCRs. However, the intracellular signaling pathways triggered by C5aR on immune-effector cells are not well understood. In this report we present data showing that, in human monocyte-derived macrophages, C5aR uses the intracellular signaling molecule sphingosine kinase (SPHK)1 to trigger various physiological responses. Our data show that C5a rapidly stimulates the generation of sphingosine-1-phosphate, SPHK activity, and membrane translocation of SPHK1. Using an antisense oligonucleotide against SPHK1, we show that knockdown of SPHK1 abolishes the C5a-triggered intracellular Ca(2+) signals, degranulation, cytokine generation, and chemotaxis. Our study shows for the first time that SPHK1 not only plays a key role in the generation and release of proinflammatory mediators triggered by anaphylatoxins from human macrophages but is also involved in the process of immune cell motility, thus pointing out SPHK1 as a potential therapeutic target for the treatment of inflammatory and autoimmune diseases.     

Aptamers that bind to the human complement component receptor hC5aR1 interfere with hC5aR1 interaction to its hC5a ligand

The complement system plays an important role in inflammation and immunity. In this system, a potent inflammatory ligand is C5a, which initiates its effects by activating its core receptor C5aR1. Thus, compounds that interfere with the C5a–C5aR1 interaction could alleviate some inflammatory conditions. Consequently, several ligands that bind to either C5a or C5aR1 have previously been isolated and evaluated. In the present study, two RNA aptamers, aptamer 1 and aptamer 9, that specifically bind to hC5aR1 with much higher affinity than antibodies were isolated. These two aptamers were tested for their ability to interfere with the cognate ligand of hC5aR1, C5a, using a chemotaxis assay. Both aptamer 1 and 9 interfered with the C5a interaction, suggesting that the aptamers recognized the extracellular domain of hC5aR1 responsible for hC5a ligand binding. Considering the higher affinity of aptamers to the hC5aR1 and their interference with hC5a ligand binding, further study is warranted to explore not only their applications in the diagnosis of inflammatory diseases but also their usefulness in modulating hC5a and hC5aR1 interactions.     

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.     

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function: IMPLICATIONS FOR AN ACTIVATION MICROSWITCH REGION*

The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined by three intra- and three extracellular loops, whose local conformations are important in receptor signaling. Many 7TM receptors contain a cysteine residue in the third extracellular loop (EC3) and a complementary cysteine residue on the N terminus. The functional role of such EC3-N terminus conserved cysteine pairs remains unclear. This study explores the role of the EC3-N terminus cysteine pairs on receptor conformation and G protein activation by disrupting them in the chemokine receptor CXCR4, while engineering a novel EC3-N terminus cysteine pair into the complement factor 5a receptor (C5aR), a chemo attractant receptor that lacks it. Mutated CXCR4 and C5aRs were expressed in engineered yeast. Mutation of the cysteine pair with the serine pair (C28S/C274S) in constitutively active mutant CXCR4 abrogated the receptor activation, whereas mutation with the aromatic pair (C28F–C274F) or the salt bridge pair (C28R/C274E), respectively, rescued or retained the receptor activation in response to CXCL12. In this context, the cysteine pair (Cys³⁰ and Cys²⁷²) engineered into the EC3-N terminus (Ser³⁰ and Ser²⁷²) of a novel constitutively active mutant of C5aR restrained the constitutive signaling without affecting the C5a-induced activation. Further mutational studies demonstrated a previously unappreciated role for Ser²⁷² on EC3 of C5aR and its interaction with the N terminus, thus defining a new microswitch region within the C5aR. Similar results were obtained with mutated CXCR4 and C5aRs expressed in COS-7 cells. These studies demonstrate a novel role of the EC3-N terminus cysteine pairs in G protein-coupled receptor activation and signaling.