Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a...

Characterization of a C3a Receptor in Rainbow Trout andXenopus: The First Identification of C3a Receptors inNonmammalian Species1

Hani Boshra,2* Tiehui Wang,2† Leif Hove-Madsen,2‡ John Hansen,2§ Jun Li,2*Anjun Matlapudi,* Christopher J. Secombes,† Lluis Tort,¶ and J. Oriol Sunyer3*

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. BecauseC3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fishimplied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aamolecule (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 immu-nofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesserextent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in troutleukocytes. 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 extrapiece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aRcharacterized 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. The Journal of Immunology, 2005, 175: 2427–2437.

A ctivation of the complement system results in the gen-eration of anaphylatoxin molecules C3a, C4a, and C5a(1). In mammals these molecules are considered to be

endogenous danger signals that induce the development of an in-flammatory response and trigger the activation of several key in-nate immune processes. All three anaphylatoxins share a high de-gree of homology and have been found to possess overlappingfunctions. The C5a anaphylatoxin is considerably more potent thanC3a and C4a in inducing biologically relevant responses (2, 3).The role of C4a in inflammation is speculative to date. Commonfunctions of C3a and C5a in mammals include their ability to in-duce chemotaxis, respiratory burst, and the expression of severalproinflammatory cytokines in a variety of leukocytes (4–7). Themajor known tasks attributed solely to C3a involve the induction

of chemotaxis specifically in eosinophils and mast cells as well asthe inhibition of the polyclonal Ab response (8–12). In the lastyear, several reports have demonstrated additional important rolesof C3a in innate and adaptive immunities. In this regard, a newstudy demonstrates that C3a has previously unforeseen antibacte-rial properties (13). Another report has shown that human mono-cyte-derived dendritic cells can be chemoattracted to C3a afterup-regulation of the C3aR with IFNs (14). Recent studies havedemonstrated a novel role of the C3aR in the retention of hemo-poietic stem/progenitor cells in bone marrow (15). The role of C3ain adaptive immunity has been demonstrated recently in a studyshowing that C3a down-regulates the Th2 response to epicutane-ously introduced Ag (16). Thus, it is becoming apparent thatthe functions of C3a in immunity are greater than previouslyanticipated.

C3a and C5a elicit their biological activities through binding toC3aR and C5aR, respectively. These two receptors are members ofthe rhodopsin family of G protein-coupled receptors, and they havebeen characterized in a variety of mammalian species, includinghumans (17, 18), rat (19, 20), dog (21), mouse (22, 23), and guineapig (19, 24). A C5a-like receptor (C5L2) recently characterized inmice and humans has been shown to bind C5a and its desarginatedderivative (C5adesArg). In contrast to C5aR, C5L2 appears to beuncoupled from heterotrimeric G proteins. Because C5L2 seems tolack the capacity to transduce signals, it has been suggested that itacts as a decoy receptor, thereby modulating the concentration ofboth C5a and C5adesArg (25, 26). C3aR and C5aR have a widecellular distribution and have been shown to be expressed in cellsof myeloid and nonmyeloid origin (27–35).

In mammals, the C3aR is the only member of the rhodopsinfamily of seven-transmembrane, G protein-coupled receptors withan unusually large second extracellular loop (ECL2) between thefourth and fifth transmembrane regions (TM4 and TM5) (3). It is

*Department of Pathobiology, University of Pennsylvania School of Veterinary Med-icine, Philadelphia, PA 19104; †Scottish Fish Immunology Research Center, Univer-sity of Aberdeen, Aberdeen, Scotland; ‡Cardiology Department, Hospital de Sant Pau,Barcelona, Spain; §Western Fisheries Research Center, Seattle, WA 98115; and ¶Unitof Animal Physiology, Department of Cell Biology, Physiology, and Immunology,Universitat Autonoma de Barcelona, Bellaterra, Spain

Received for publication January 20, 2005. Accepted for publication April 15, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Science Foundation Grant MCB-0417078, theNational Research Initiative of the U.S. Department of Agriculture Cooperative StateResearch, Education and Extension Service, Grant 2004-01599; a contract from theEuropean Community (Q5RS-2001-002211); and Grant AGL2000-0349 from theMinisterio de Ciencia y Tecnologı́a.2 H.B., W. T., HM. L., H.J., and L.J. contributed equally to this study.3 Address correspondence and reprint requests to Dr. J. Oriol Sunyer, Department ofPathobiology, University of Pennsylvania School of Veterinary Medicine, 413Rosenthal, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address:[email protected]

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

worth noting that there appears to be very little sequence homol-ogy of this loop among species. Interestingly, studies of guinea pigC3aR have identified two alternatively spliced receptors, lacking34 residues of the large ECL2. No functional differences could befound in the expressed guinea pig spliced C3aR products (36).

Little is known about the evolution of C3aR and C5aR. How-ever, anaphylatoxin activity has been demonstrated in primitiveinvertebrate species, where C3a-like peptides have been shown toinduce hemocyte chemotaxis in tunicates, thereby implying thepresence of a C3a-like receptor in these animals (37, 38). A recentreport in trout showed for the first time in teleosts the presence ofthree C3a molecules generated from three trout C3 isoforms (C3-1,C3-3, and C3-4). Each of the three C3a isoforms stimulated to asignificant degree the respiratory burst of trout head kidney leu-kocytes, suggesting that teleost fish contain a C3aR (39). Recently,recombinant trout C5a has been produced and shown to play aprominent role in inducing leukocyte chemotaxis (40, 41) and re-spiratory burst (40). Thereafter, a C5aR was identified in rainbowtrout, representing the first cloned (42, 43) and functionally char-acterized anaphylatoxin receptor in a nonmammalian species (42).It is well known that C3aR and C5aR share a high degree of ho-mology, to the extent that it has been hypothesized that both re-ceptors represent the duplication products of a single ancestral re-ceptor. Thus, the presence of a bona fide C5aR in teleosts isrelevant because it may be indicative of the existence of C3aR inthese animal species. Thus, this study was initiated to explore theabove-mentioned hypothesis with the idea of finding a homologueof C3aR in rainbow trout. In this study we report the character-ization of a trout molecule whose primary structure appears to behighly similar to that of mammalian C3aRs (MC3aRs).4 Howeverthis trout C3a-like receptor (TC3aR) was found to lack a signifi-cant portion of the large extracellular loop between TM4 and TM5characteristic of MC3aRs, which is substituted instead by a muchsmaller loop. We also demonstrate the ability of this trout receptorto interact with trout C3a.

Materials and MethodsFish

Rainbow trout (100–200 g) were obtained from Limestone Springs FishFarm. Fish were maintained in aquarium tanks using a water recirculationsystem involving extensive biofiltration, UV sterilization units, and ther-mostatic temperature control. Water temperature was maintained continu-ously at 12–14°C.

cDNA cloning of TC3aR and sequence phylogenetic analysis

Trout cDNA was generated from trout liver as previously described (42)using an Oligotex Direct mRNA kit (Qiagen), according to the manufac-turer’s recommendations. mRNA (2.0 �g) was reverse transcribed to neg-ative strand cDNA with oligo(dT) (0.05 �g/�l) and 40 U of SuperScriptreverse transcriptase II (Invitrogen Life Technologies) for 1 h at 42°C. Aprimer set was designed on the basis of a 729-bp established sequence tag(EST; GenBank accession no. CA373689) from rainbow trout (Oncorhyn-chus mykiss) that was found to be homologous in sequence to mammalianC3aRs. The forward primer (5�-TCAAAGATGGGGGACAACA-3�) andthe reverse primer (5�-CAGACAGCGATGACCAGTA-3�) were used togenerate a 713-bp DNA fragment using the proofreading enzyme PfuTurbo (Stratagene) according to the manufacturer’s recommendations andwith the following thermocycling conditions: 95°C for 2 min; 40 cycles of95°C for 30 s, 55°C for 1 min, and 72°C for 1 min; and 72°C for 10 min.This fragment was also used as a probe for Northern and Southern blotting.PCR products were cloned into a TOPO zero blunt vector (Invitrogen LifeTechnologies) and sequenced with a 3100 DNA analyzer (Applied Bio-

systems). Consensus sequences were generated from comparisons of re-peated amplifications from trout liver mRNA using SeqMan and MegAlign(DNA Star) software. The full-length cDNA of TC3aR was obtained byperforming 5� and 3� RACE using SMART cDNA prepared from livertissues, as described by Wang et al. (44). 3� RACE using a forward primer(5�-GGGACAACATGGATTTCTCAG-3�) based on our initial TC3aREST yielded a 1463-bp fragment. When sequenced, the 3� end was foundto possess a stop codon, a 3�-untranslated region, and a poly(A) tail. 5�RACE was performed using a primer corresponding to the 3� untranslatedregion (5�-CCAACAGCTTTACACAAAACGCCATC-3�) and yielded anadditional 13 bp located 5� to the original EST. Sequence alignments andthe phylogenetic tree were generated using the Clustal X software package(45). TM regions in TC3aR were predicted using TMpred software (46).

Northern blotting

TRIzol reagent (Invitrogen Life Technologies) was used to obtain the totalRNA from various tissues and leukocytes of rainbow trout. RNA was quan-tified, and 10 �g/lane was size-fractionated on agarose-formaldehyde gelsand transferred to nylon-supported nitrocellulose membranes (Bio-Rad) bycapillary blotting. The blot was thereafter exposed to UV cross-linking tofix the RNA to the membrane. The 713-bp cDNA corresponding to ourinitial TC3aR EST was gel purified and radiolabeled with [32P]dCTP usingthe Ready-to-Go labeling system (Amersham Biosciences) and was puri-fied using ProbQuant G-50 microcolumns (Amersham Biosciences). Theblot was prehybridized in warm Express hybridization solution (BD Clon-tech) at 68°C for 30 min in a hybridization oven (Problot 6; Labnet). Theblot was hybridized at 68°C for 60 min in Express hybridization solutionwith 1–2 � 106 cpm of labeled probe/ml. After hybridization, the blot wasrinsed three times in 2� SSC with 0.1% SDS for 30 min at room temper-ature, then washed in 0.1� SSC with 0.1% SDS with continuous agitationat 50°C for 40 min. After washing, the blot was exposed to x-ray film(Kodak XB; Eastman Kodak) in an autoradiography cassette (Fisher Sci-entific). The expression of TC3aR RNA was normalized for equal loadingand transfer to 28S RNA.

Southern blotting

Genomic DNA (10 �g) isolation and blotting procedures have been pre-viously described (42). For Southern blotting, the 713-bp cDNA fragmentcorresponding to our initial EST was generated by PCR. The gel-purifiedPCR product was then randomly labeled (Amersham Biosciences) with[32P]dCTP and used as a probe (65°C) under stringent conditions (0.25�SSC/0.25% SDS, 64°C final wash). The blot was exposed to film for6 days.

Generation of Abs against TC3aR

A 20-aa peptide corresponding to the N-terminal region of TC3aR (EHYG-NFSENYVTESYGEFDC) was synthesized by Biosynthesis. Matrix-as-sisted laser desorption mass spectrometry was used to determine the purityof the peptide. The synthesized peptide was coupled to keyhole limpethemocyanin by the glutaraldehyde method and was used to raise Abs inrabbits (Biosynthesis). Ab titers were determined by ELISA. The Ig frac-tion of the antiserum was first purified using a HiTrap protein G columnaccording to the instructions of the manufacturer (Amersham Biosciences).Thereafter, specific Abs against the TC3aR peptide were purified by af-finity chromatography using the synthetic peptide coupled to cyanogenbromide-activated Sepharose (Amersham Biosciences).

Isolation of PBLs and head kidney leukocytes (HKLs)

PBLs were isolated as previously described (42). Briefly, blood was col-lected from trout through the caudal vessel using a heparinized syringe anda 21-gauge needle. After extraction, the blood was immediately diluted(1/5) with EMEM (American Type Culture Collection) supplemented with100 U/ml penicillin, 100 �g/ml streptomycin, and 25 U/ml heparin, thenplaced on ice. The blood cell suspension was thereafter layered onto a51/34% discontinuous Percoll (Sigma-Aldrich) density gradient and cen-trifuged at 400 � g for 30 min. The band of cells lying at the interface wascollected, and the cells were washed with HBSS or kept in EMEM sup-plemented with 100 U/ml penicillin and 100 �g/ml streptomycin. HKLswere isolated as previously reported (39).

Flow cytometric analysis

Flow cytometric analysis of PBLs and HKLs was performed using condi-tions previously described (42). Briefly, 1 � 106 cells were suspended in1� PBS/2% FCS and incubated for 30 min at room temperature withaffinity-purified rabbit anti-TC3aR, alone or in combination with anti-trout

4 Abbreviations used in this paper: MC3aR, mammalian C3aR molecule; ECL2, sec-ond extracellular loop; [Ca2�]i, intracellular calcium; EST, established sequence tag;HKL, head kidney leukocyte; ICD, intracellular domain; IF, immunofluorescence;PKC, protein kinase C; TC3aR, trout C3aR; XC3aR, Xenopus C3aR; TM, transmem-brane region.

2428 CLONING AND CHARACTERIZATION OF FIRST NONMAMMALIAN C3aR

IgM (mAb 1.14 provided by Dr. G. Warr, Medical University of SouthCarolina, Charlestown, SC) or anti-trout thrombocyte (mAb28.D7) mAbs.As a negative control, cells were incubated with Abs purified from preim-munized rabbit serum or control mouse IgG. After two washes in PBS/2%FCS, cells were stained with either FITC-conjugated anti-rabbit IgG orallophycocyanin-conjugated anti-mouse IgG. Cells were washed twomore times, and cell cytometric analysis was performed using a standardFACScan (BD Biosciences). For each sample, 20,000 individual cells were

analyzed, and the resulting data were analyzed using the programCellQuest (BD Biosciences).

Indirect immunofluorescence microscopy

Indirect immunofluorescence microscopy was performed using cellsstained with anti-TC3aR in a similar manner as described for cell cyto-metric assays, with an additional step of incubation with Hoechst stain

FIGURE 1. Amino acid comparisons of TC3aR and XC3aR with MC3aR sequences. Putative TMs are overlined. Extracellular and intracellular domainsare indicated by upward and downward arch symbols, respectively. Residues important for C3aR function are shown in bold and include the following:Tyr200 (trout numbering) and the PKC recognition domain in the trout sequence (FKSQRA). The serine/threonine C-terminal phosphorylation sites in troutand Xenopus are also in bold (see Results and Discussion). Predicted N-glycosylation sites are circled. Asterisks denote identities in all sequences, and gapsare denoted by dashes. Colons indicate strongly similar amino acids, whereas single dots infer weakly similar residues. The N and C termini are indicatedby lines linked to TM1 and the C-terminal valine of the trout sequence, respectively. The peptide sequence used to generate polyclonal Abs (at the Nterminus of the trout sequence) is boxed.

2429The Journal of Immunology

(according to the manufacturer’s recommendations) to visualize the cellnucleus.

Generation of TC3a and C3adesArg

TC3a was generated as previously described (39) from C3-1, the mostactive and abundant TC3 isoform (47). Briefly, trout C3-1, Bf/C2, andfactor D molecules were purified from trout serum as described previously(47, 48). To produce the C3a anaphylatoxin, purified TC3-1 (5 mg), troutfactor B/C2 (250 �g), and trout factor D (30 �g) were incubated for 30 minat room temperature in the presence of 5 mM Mg2� in PBS. To purify theC3a fragment, the total reaction mixture (0.8 ml) was passed through aSuperdex 200 gel filtration column (Amersham Biosciences) equilibratedwith PBS, pH 7.4. The purity of the C3a was determined by SDS-PAGEand N-terminal sequencing. In mammals, the C-terminal Arg of C3a iscritical for several of its functions, including its ability to induce intracel-lular calcium mobilization in leukocytes and other cell types. To removethe C-terminal Arg from the TC3a, this anaphylatoxin was treated withcarboxypeptidase B (20%, w/w) for 1 h at room temperature, then purifiedon a Superdex 75 gel filtration column (Amersham Biosciences) equili-brated with PBS, pH 7.4 (39).

Measurement of intracellular calcium [Ca2�]i mobilization byconfocal microscopy

Trout leukocytes (HKLs) were incubated with the acetoxymethyl ester ofthe fluorescent calcium indicator fluo-3 (5 �M) for 15 min. Cells were thenwashed twice with Tyrode’s solution and left on coverslips for cell attach-ment and de-esterification of fluo-3 for at least 30 min before experimentswere begun. Fluorescence was measured using a confocal microscope(Leica SP2; AOBS) with the excitation beam at 488 nm attenuated to 2.5%.Fluorescence emission was collected between 500 and 650 nm. Experi-ments were begun by flushing the cells with control Tyrode’s solution toremove debris and nonsticking cells. Baseline fluorescence was recorded inthe control solution for at least 10 min before C3a (5 nM) was added aloneor in combination with anti-TC3aR IgG or the control preimmune IgG. Todetermine the time course of the action of C3a with or without Ab, fluo-rescence was measured in 10–15 randomly selected cells every 30 s overa 15-min period. The fluorescence intensity was expressed as a percentageof the baseline fluorescence (control) before addition of C3a with or with-out Ab, and the peak fluorescence was measured after addition of C3aalone, C3a (5 nM) with the control preimmune IgG (40 nM), or C3a withanti-TC3aR IgG (40 nM). For statistical evaluation of the results, valuesfrom each animal were averaged, giving one value for each condition peranimal. Statistical analysis was performed on the average values from eachanimal, with n representing the number of animals. Student’s t test for

unpaired samples was used, and differences were considered statisticallysignificant at p � 0.05.

ResultsIsolation and sequence analysis of TC3aR cDNA

We originally identified a 729-bp EST from rainbow trout thatshowed a high degree of homology to mammalian C3aR. Using 5�and 3� RACE, we obtained a 1476-bp product (TC3aR) that in-cluded an initiation site, an open reading frame encoding for 364aa, and a 3� polyadenylation signal. When aligned with C3aR fromhuman, mouse, rat, and guinea pig, a significant degree of homol-ogy was observed throughout the molecule (Fig. 1), with one ex-ception: a region spanning �137 aa in MC3aRs was noticeablyabsent in TC3aR. Upon additional analysis, it was shown that thisportion of sequence corresponded to a large segment of the ECL2of mammalian C3aR (represented in Fig. 2). C3aR in mammals isthe only rhodopsin receptor known to have such a large ECL2 (49,50). In the alignment of Fig. 1, we also included a sequence fromXenopus tropicalis (a diploid frog) highly homologous to TC3aRand MC3aR. Like TC3aR, the Xenopus molecule (XC3aR) lackeda large portion of the ECL2 (113 residues), although this regioncontained 24 more residues than the ECL2 of TC3aR. The XC3aRsequence was found after a multiple alignment analysis of TC3aRand MC3aRs sequences with available Xenopus genomic scaffoldslocated at the Ensembl Genome Browser web site of the WellcomeTrust Sanger Institute (�www.ensembl.org/�). More excitingly, insilico analysis of the Xenopus scaffold containing the XC3aR geneshowed that XC3aR and MC3aR genes were located in syntenicregions, further supporting designation of the trout and Xenopusmolecules as C3aR (Fig. 3). We were unable to find the corre-sponding syntenic region in the genome of zebrafish, although wedid find a molecule highly homologous to TC3aR and XC3aR in adifferent region of the zebrafish genome (data not shown). Thislack of synteny in regions of the genome between the zebrafish andXenopus (or mammals) has been observed for other genes (i.e.,genes within the MHC region (51)). It should be stressed thatexhaustive in silico analysis of the genomes of Xenopus or ze-brafish did not yield a C3aR-like molecule with an extra largeECL2 similar in size to that of mammals. Similarly, analysis of thetrout or salmon ESTs (�244,837 ESTs) deposited at the NationalCenter for Biotechnology Information or at the Institute forGenomic Research did not yield either a C3aR-like molecule withan extra large ECL2. All the above results combined suggest thatC3aR in fish and amphibians lacks the additional extra piece ofsequence that is uniquely present in the ECL2 from all MC3aRmolecules.

Hydropathy analysis confirmed that TC3aR and XC3aR did pos-sess seven-transmembrane domains normally associated withC3aR (and all rhodopsin receptors) in higher vertebrates (Fig. 4).As stated above and shown in Figs. 1 and 2, there was a noticeabledifference in the amount of residues of ECL2 between TM4 andTM5 of TC3aR and XC3aR compared with the same region ofMC3aR molecules. In humans, the entire ECL2 contains 175 aa(50), whereas in TC3aR and XC3aR, the area spanning the ECL2is significantly shorter, comprising 36 and 62 aa, respectively (Fig.2). It is worth noting that MC3aR is fully functional even afterdeletion of 65% of the residues of ECL2 (52). In fact, mutagenesisstudies have shown that the crucial residue (Tyr174 in humans)required for the interaction of C3aR with C3a is located at thebeginning of the ECL2 (53). Significantly, this critical residue isconserved in the ECL2 of TC3aR and XC3aR. When aligned withother MC3aRs, this tyrosine is located two amino acids down-stream from Cys172 (human numbering). In TC3aR and XC3aR, the

FIGURE 2. Schematic structure of C3aR showing the different sizes ofthe ECL2 in trout, Xenopus, and human C3aR. The ECL2 of C3aR isshown between TM4 and TM5. Numbers in parentheses indicate theamount of residues within the ECL2.


corresponding tyrosine is also two residues from Cys198 (trout num-bering). It should be noted that mammalian and trout C5aR also pos-sess a homologous tyrosine, but in both cases this residue is locatedfour amino acids downstream from their respective cysteines.

To find meaningful percentages of amino acid identities be-tween TC3aR and the mammalian and Xenopus C3aR sequences,the sequence alignments were performed excluding the extra �137residues of the ECL2 from MC3aRs or excluding the extra 26residues of the ECL2 from XC3aR lacking in TC3aR. The analysisshowed that TC3aR presented a significantly greater degree of ho-mology to C3aR than to C5aR molecules or other members of therhodopsin gene family. Thus, TC3aR showed 40% identity toXC3aR, 38.3% identity to murine C3aR, 34.0% identity to humanorphan receptor ChemR23, 32% identity to human formyl peptidereceptor 1, 29.8% identity to TC5aR, and 25.3% identity tomurine C5aR.

Further analysis of the structure of TC3aR and XC3aR indicatedthat similar to MC3aRs and MC5aRs, these sequences possessed aserine/threonine-rich C terminus in which these residues may rep-resent phosphorylation sites that become modified as a result ofligand stimulation (36). Conservation of post-translational modi-fications, including N-linked glycosylation, was also observed. Todate, all characterized MC3aRs have been found to possess threeto eight glycosylation sites, in contrast with the one or two con-tained in C5aR molecules. It is interesting that one to four (de-pending on the species) of these glycosylation sites are localized inthe ECL2 (36). TC3aR and XC3aR were both found to possessfour potential glycosylation sites (Fig. 1). Despite their consider-ably shorter ECL2, both TC3aR and XC3aR still contained twoN-linked glycosylation sites within that region. This observation issignificant, because it indicates a higher degree of conservation of

TC3aR to C3aR rather than to MC5aR, in which the ECL2 isdevoid of glycosylation.

In our previous characterization of TC5aR, we found that TMsamong C5aR molecules were more highly conserved than theirintra/extracellular regions (42). This does not appear to be the casewith TC3aR. Although TM2 and TM3 showed the highest degreeof conservation among all C3aR TMs (61.5 and 68.2%, respec-tively), there also existed a high degree of conservation of all threeC3aR intracellular domains (ICD), with sequence identity valuesranging from 46.7% (ICD3) to 85.7% (ICD1). The extracellulardomains, especially the N terminus and ECL2 regions, remainedthe least conserved of the receptor, with �6% sequence identity.

It has been hypothesized that C3aR serves as a putative substratefor protein kinase C; in all MC3aRs, this motif has been found tobe conserved as XKSXXKX (36). Although TC3aR does not pos-sess this exact motif, it contains a sequence signature that is con-sistent with protein kinase C (PKC) recognition (FKSQRA) (54),which is also located in IC3. PKC recognition domains analogousto those mentioned above in mammalian and trout C3aRs wereabsent in the IC3 of all cloned C5aR, including TC5aR.

In Fig. 5, a phylogenetic tree was constructed using TC3aR,XC3aR, MC3aR, C5aR, TC5aR, along with other mammalian rho-dopsin receptors. Both trout and Xenopus C3aR molecules clus-tered with the MC3aR molecules (Fig. 5). The tree composite alsosuggests that TC3aR is the most ancestral of all C3aR molecules.

Northern blot analysis of TC3aR

Total RNA was obtained from a variety of trout tissues and leu-kocytes and was separated by formaldehyde agarose gel electro-phoresis. After transfer to nylon membranes, TC3aR RNA expres-sion was detected using a 713-bp P32 labeled probe, corresponding

FIGURE 3. The genomic regions containing Xenopus and human C3aR are syntenic. The upper part of the figure represents genomic scaffold 811 fromXenopus (X). The scaffold was found at the Ensembl Genome Browser web site of the Wellcome Trust Sanger Institute (�www.ensembl.org/�). The scaffoldspans a genomic fragment of 500 kb. XC3aR is contained within bases 120,206–122,817. The entire scaffold was found to be syntenic with the region fromhuman chromosome 12 that contains the human C3aR gene (H; lower part of the figure). This region spans from 6.8 to 8.2 Mb of human chromosome12. The lines connecting the human and Xenopus genomic regions link the gene orthologs between the two species. Below the gene name, is indicated eitherthe GeneScan number (upper part; Xenopus scaffold) or the Ensembl gene identification number (lower part; human chromosome 12 region). The graphicindicates the order in which the genes are positioned in the genomic regions of Xenopus and human; however, the distances from gene to gene are notproportional.


to our original TC3aR EST. In all samples, no more than one bandwas observed, which was estimated to be �2.4 kb (Fig. 6). Inmammals, C3aR mRNA has been detected at sizes ranging from2.1 kb (human and mouse) (17, 22) to 3 kb (guinea pig) (36).Normalization of the TC3aR signal with 28S rRNA indicated thatexpression was strongest in blood leukocytes. A significant level ofTC3aR message was found in the rest of the samples tested withina relatively short period of exposure (1 day). It is difficult to statewhether this expression was due to unavoidable blood contamina-tion of the sampled organs or was the true expression of TC3aR inthese tissues. In this regard, the expression of TC3aR in gills (anorgan rich in blood leukocytes) was found to be comparable to thatof PBLs (data not shown).

Southern blot analysisAs was the case for Northern blot analysis, the 713-bp probe waslabeled with 32P and used as a probe for Southern blotting. Thisprobe was used because analysis of genomic DNA by PCR indi-cated that no introns were present within this fragment. Each of thethree restriction enzymes yielded two or three different digestionproducts (Fig. 7). Fish 2 and 3 showed additional bands in the blot,

providing evidence of allelic variation in these animals. Takinginto account that no restriction sites exist within the probe for theenzymes used in the digestion, the Southern blot data appear tosuggest that two TC3aR genes exist in the trout genome. The laterwas almost expected due to the quasi-tetraploid nature of rainbowtrout. It should be pointed out, however, that screening of troutliver and head kidney libraries by RT-PCR or colony blotting us-ing the 713-bp EST fragment failed to yield other variants or iso-forms of TC3aR. Moreover, the Northern blot analysis detectedonly a single band. Significantly, multiple sequence alignmentanalysis of our TC3aR sequence with all ESTs comprised at theInstitute for Genomic Research and National Center for Biotech-nology Information Unigene EST indexes (�155,000 trout ESTs)did not yield any EST significantly similar in primary and second-ary structures to TC3aR. Although not definitive, these facts sug-gest that only one of the two TC3aR genes is expressed.

Binding of anti-TC3aR to PBLs

Polyclonal Abs were generated against a 20-aa peptide corre-sponding to a portion of the putative N-terminal extracellularregion of TC3aR (boxed residues in Fig. 1). Anti-peptidespecific Abs were affinity purified using a column to which thepeptide had been coupled. When used for flow cytometricanalysis, �83% of all PBLs were stained with the Ab, asindicated in the shift of fluorescence shown in the histogram inFig. 8A. Incubation of the Ab preparation with a molar excessof the TC3aR peptide inhibited �90% of the Ab staining ofPBLs, providing additional evidence of the specificity of the Ab(data not shown). As shown in Fig. 8, staining was localized totwo distinct cell populations, designated R1 and R2. The cellsin R1 (�51% of the PBLs), displayed low forward and sidescatters and showed the strongest staining. The R2 population(�25% of the PBLs) exhibited the highest forward and sidescatters (composed of granulocytes in trout), although theystained to a lesser degree compared with the R1 population.Granulocytes in mammals have also been shown to expressC3aR (35, 55). The R3 population (�17% of the PBLs) repre-sented the negative cells, because these cells displayed the samefluorescence intensity as those stained with the preimmunepolyclonal rabbit IgG (Fig. 8D). Costaining analysis using theanti-TC3aR and an anti-trout thrombocyte mAb showed that�95% of the TC3aR-negative cells (shown in R3, Fig. 8D)were, in fact, thrombocytes (data not shown). As expected, theanti-TC3aR did not stain trout erythrocytes (data not shown).The fact that the anti-TC3aR did not stain thrombocytes anderythrocytes supports the specificity of the anti-TC3aR and is inagreement with the lack of staining of thrombocytes and RBCsin humans when using anti-human C3aR (55).

Costaining analysis using the anti-TC3aR in combinationwith a mAb specific for trout IgM (mAb 1.14) that stains Blymphocytes in trout (56, 57) showed the presence of TC3aR inall B cells (Fig. 8F). This pattern was displayed in all fishanalyzed (n 8). The double-positive cells (representing�36% of the PBLs) displayed the same low forward and sidescatter properties of the R1 population in Fig. 8 (data notshown). This was expected, because B cells are small agranularcells. It should be noted that this double-positive populationshowed some variability among different individuals, rangingfrom �29 to 55% of the PBLs analyzed.

Trout HKLs displayed a very similar staining pattern, in whichB cells showed the highest binding to anti-C3aR, and �95% ofgranulocytes stained, although once again, to a lesser extent (datanot shown).

FIGURE 4. Prediction of TM regions of TC3aR, XC3aR, and humanC3aR. TM plotting of C3aR amino acid sequences from trout (upperpanel), Xenopus (middle panel), and human (lower panel) were obtainedusing the TMpred program, based on the method of Hofman and Stoffel(46). TM domains 1–7 and ECL2 are also indicated.


Immunofluorescence (IF) microscopy

IF microscopy using the affinity-purified Abs indicated that TC3aRis expressed on the cell surface of trout granulocytes and lympho-cyte-like cells of PBLs (Fig. 9) and HKLs (data not shown), andthat the staining pattern was punctuated and patchy (Fig. 9). Inagreement with the flow cytometric results, it could be observedthat in lymphocytes the patchy areas were generally more abun-dant than in granulocytes (Fig. 9). A similar scattered pattern ofC3aR staining has been shown on human PMN (55) and astrocytes(34, 58).

Inhibition of C3a-mediated intracellular calcium [Ca2�]i

mobilization by anti-TC3aR Abs

In mammals, it is well known that C3a induces increases in[Ca2�]i in a variety of cells (59 – 62). We determined firstwhether TC3a could have a similar effect on trout leukocytes,then we investigated the ability of the anti-TC3aR to inhibitpotential C3a-mediated increases in [Ca2�]i. To evaluate the[Ca2�]i-mobilizing capacity of TC3a, its effect on fluo-3 fluo-rescence was measured, as shown in Fig. 10. Because analysis

of [Ca2�]i using confocal microscopy have never been per-formed with trout leukocytes, we first optimized the experimen-tal conditions to obtain stable baseline fluorescence measure-ments over time. This led to measurements in which the averagebaseline fluorescence in control cells increased only by 1.5 2.1% over a 10-min period (85 cells from six trout). The middlepanel of Fig. 10A shows one example of fluorescence fromcontrol cells after 10-min exposure to control Tyrode’s buffer.Most of the cells showed no or very little fluorescence. In theright panel of Fig. 10A, the increase in cell fluorescenceinduced by exposure to TC3a (5 nM) is clearly shown. Asobserved in that figure, a majority of the cells were stimulated(turned to green) by C3a, representing 68 4% of the cellsexamined (59 cells from a total of three fish). The left panel in

FIGURE 5. Phylogenetic tree of TC3aRand other related members of the rhodopsingene family. Amino acid alignments were per-formed using Clustal X, which was used togenerate an unrooted neighbor-joining tree.Numbers on the branches indicate total recov-ery from 1000 bootstrap replications. The phy-logenetic tree was created using alignmentsperformed with the entire mammalian C3aRsequences. Accession numbers used to con-struct the tree are as follows: TC5aR,AY438032; human C5aR, NP_001727; guineapig C5aR, O70129; rabbit C5aR, Q9TUE1; ratC5aR, NP_446071; mouse C5aR, AAB97774;dog C5aR, P30992; human C5L2, BAA95414;mouse C5L2, BAC35303; TC3aR, AJ616902;human C3aR, AAH20742; guinea pig C3aR,O88680; rat C3aR, O55197; mouse C3aR,AAH03728; mouse Dez, NP_032179; humanChemR23, CAA75112; human formyl peptidereceptor 1 (FPR1), P21462; human FPR1,NM_002029; mouse FPR1, NP_038549; andmouse FPRL1, NP_032068.

FIGURE 6. Tissue-specific expression of TC3aR by Northern blot. Tenmicrograms of total RNA from the indicated tissues of rainbow trout waselectrophoresed, blotted onto a nitrocellulose membrane, and hybridizedwith a DNA probe corresponding to a 713-bp fragment of TC3aR.Ethidium bromide-stained 28S is shown as a loading control.

FIGURE 7. Southern blot analysis. Genomic DNA from four individualrainbow trout was digested with EcoRI, EcoRV, and HindIII. The blot wasthen hybridized with a DNA probe corresponding to a 713-bp fragment ofTC3aR. Values on the right of the blot indicate kilobases.


Fig. 10A shows a transmission (brightfield) image of the mi-croscope field with the cells selected for fluorescence analysis.Fig. 10B shows the time course of the increase in fluo-3fluorescence after addition of 5 nM C3a. To verify that theeffect of C3a was specific, the action of the desarginated formof C3a (C3adesArg) on [Ca2�]i mobilization was also analyzed.In contrast to C3a, C3adesArg did not increase [Ca2�]i in troutleukocytes, suggesting that the stimulatory effect was a directeffect of C3a (Fig. 10C). This result is in agreement with thesituation in mammals, in which C3adesArg does not have aninfluence on [Ca2�]i mobilization (3, 63).

To verify that the cloned trout receptor (TC3aR) has the capac-ity to interact with the C3a ligand, we investigated whether theanti-TC3aR could block the C3a-mediated increase in [Ca2�]i thatis shown in Fig. 10C. To this end, cells were exposed to 5 nM C3ain the presence or the absence of an 8-fold excess of the anti-TC3aR IgG. Preimmune IgG was used as a control. Fig. 10Cshows that the anti-TC3aR almost completely abolished the stim-ulatory effect of C3a in inducing increases in [Ca2�]i, whereaspreimmune IgG had no effect. Thus, the inhibitory action of theanti-TC3aR in C3a-mediated increases in [Ca2�]i supports the ideathat TC3aR is a bona fide C3aR.

DiscussionOur current knowledge of the structure and function of C3aR mol-ecules comes from the study of MC3aRs, with nothing beingknown about the evolutionary origins of this important proinflam-matory molecule. Thus, up to this point, no C3aRs have been iden-tified in nonmammalian species. The present study was thereforeundertaken to identify a homologous receptor in an evolutionarilyold vertebrate species, with the goal of better understanding theimportant structural elements and functions that have been con-served throughout the evolution of this receptor.

Recent studies by us have shown that teleost fish contain C3aand C5a anaphylatoxins that play important roles in chemotaxisand respiratory burst processes, implying the presence of anaphy-latoxin receptors in these species. We (42) and others (43) haverecently reported the characterization of a bona fide TC5aR inrainbow trout. These findings suggested that the duplication eventgiving rise to C5aR and C3aR from a common ancestor might haveoccurred before the emergence of teleost fish.

In this study we have characterized a 364-residue molecule inrainbow trout that is highly homologous to all known MC3aRs.Several lines of evidence indicate that the trout molecule reportedin this study represents a true C3aR: 1) the overall primary andsecondary structures of TC3aR show a significantly higher degreeof homology to C3aR than to C5aR or other members of the largerhodopsin family of seven-transmembrane, G protein-coupled re-ceptors; 2) the phylogenetic tree composite illustrates that TC3aRclustered with XC3aR and all known mammalian C3aR molecules;3) the fact that, similar to TC3aR, the XC3aR sequence lacked alarge piece (113 residues) of the ECL2 along with evidence thatthe XC3aR gene was found to reside in a genomic region syntenicto the region containing C3aR in mammals; and 4) functional ev-idence showing that anti-TC3aR Abs inhibited C3a-mediated[Ca2�]i mobilization in trout leukocytes.

FIGURE 8. Binding of anti-TC3aR to trout PBLs. A, Histogram of cellsstained with affinity-purified rabbit anti-TC3aR (3 �g/ml), followed byFITC-conjugated goat anti-rabbit IgG (line histogram; green). As a control,cells were incubated with preimmune rabbit IgG (filled histogram; blue).B–E, Forward (FSC-H) and side (SSC-H) scatter analyses of positively (R1and R2) and negatively (R3) stained cells after incubation with anti-TC3aR. F, Double staining of trout PBLs with anti-TC3aR and anti-troutsurface IgM. Cells were costained with affinity-purified rabbit anti-TC3aR(3 �g/ml) and anti-trout IgM (3 �g/ml). One experiment of eight per-formed is shown.

FIGURE 9. Indirect IF analysis of trout PBLs with anti-TC3aR. Cellswere stained with 3 �g/ml affinity-purified rabbit anti-TC3aR (green flu-orescence) and with Hoechst staining to visualize the nucleus (blue fluo-rescence). Asterisks denote control cells incubated with preimmune rabbitIgG (3 �g/ml).


A significant feature of TC3aR was the lack of 137 residues ofthe mammalian ECL2 region, which is unusually large in MC3aRmolecules. MC3aR is the only member of the rhodopsin family ofseven-transmembrane, G protein-coupled receptors with an unusu-ally large ECL2. It is striking, however, that the only residue in theECL2 that appears to be pivotal for the interaction of C3a withC3aR (Tyr174) in humans (53) is positioned at the beginning of theloop, and it is conserved in the trout and Xenopus C3aR sequences.

Because the extra piece (137 residues) of the loop present in mam-malian ECL2 does not seem to play a role in C3a binding, it hasbeen proposed that it might bind to additional ligands and/or itmight associate with surrounding cell surface proteins (53). Com-bined with our results, these findings suggest that the ancestralmolecular architecture of C3aR did not include this extra piece ofsequence, which was probably acquired later in evolution, after theappearance of amphibians, but before the emergence of mammals.

Northern blot analysis showed that PBLs were the most plentifulsource of TC3aR message. In mammals, C3aR is expressed in awide variety of organs, although tissue distribution varies consid-erably between species. In guinea pigs, C3aR has been found to beexpressed primarily in macrophages and spleen, with residual ex-pression in liver, brain, and lung (36). However, in mice, C3aR isexpressed mainly in heart and lung tissue, with no significant ex-pression in spleen (22). This contrasts with human C3aR, which isfound to be primarily expressed in placental, heart, and lung tis-sues, with no appreciable levels found in brain (17). The highexpression of C3aR in PBLs was also confirmed at the proteinlevel, using Abs against TC3aR. Significantly, flow cytometricanalysis showed a high degree of TC3aR expression in trout Bcells, which suggests an important role for this receptor in fishimmunity. The presence of C3aR in mammalian B cells is incon-clusive at this time. Although two studies using anti-human C3aRAbs showed no C3aR staining in circulating B cells (35, 61), an-other study demonstrated the presence of C3aR at the cDNA andprotein levels in human activated-tonsil derived B cells (9). A sim-ilar situation was shown with regard to C3aR expression in humanT cells. Although unchallenged circulating T cells were shown tobe devoid of C3aR (35, 55), activated human T cells were dem-onstrated to express a functional C3aR (64). Thus, it is possiblethat the expression of C3aR in mammals depends on the activationstate of lymphocytes. Our data, however, seem to suggest that allcirculating lymphocytes in trout express TC3aR. The staining re-sults obtained for TC3aR are very similar to those previously re-ported for TC5aR (42). Like the anti-TC3aR, anti-TC5aR Abswere shown to stain all B cells as well as the granulocyte popu-lation of PBLs. In addition, anti-TC5aR, similar to anti-TC3aR,did not stain the thrombocyte population, a finding in agreementwith the lack of staining of thrombocytes in humans when usinganti-human C3aR (55). It is worth noting that although severalstudies have convincingly demonstrated that human platelets donot express C3aR (65, 66), C3a has been reported to activateguinea pig platelets (67), implying the presence of such receptorsin the platelets of these animals. It is therefore possible that inmammals, the expression of C3aR in platelets is species specific.

In conclusion, our findings represent the first structural andfunctional characterization of a C3aR in a nonmammalian species.The data presented in this study support the hypothesis that ifC3aR and C5aR diverged from a common ancestor, then this eventoccurred before the emergence of teleost fish. Given the new arrayof roles recently demonstrated for C3a and C3aR in mammals(13–16), one anticipates that the study of these molecules in fishmay identify unexpected functions of these molecules in highervertebrates.

AcknowledgmentsWe thank Xinyu Zhao (Biomedical Imaging Core, School of Medicine,University of Pennsylvania, Philadelphia, PA) for her excellent technicalassistance with the IF analysis for Fig. 9.

DisclosuresThe authors have no financial conflict of interest.

FIGURE 10. Anti-TC3aR inhibits TC3a-mediated intracellular calciummobilization in trout leukocytes. A. Trout C3a stimulates [Ca2�]i mobili-zation in leukocytes. Confocal images of Fluo-3 fluorescence in trout leu-kocytes before (control) and 10 min after exposure of the cells to 5 nMC3a. The left panel in A shows the brightfield image of the cells. Rectan-gles enclose the selected cells where changes in fluorescence were fol-lowed. B, Time course of the change in fluorescence after addition of TC3a.The panel shows the average changes in fluorescence of the total numberof cells analyzed (10–15 cells) in a representative experiment (n 3). C,An 8-fold excess of anti-TC3aR inhibits TC3a-mediated [Ca2�]i mobili-zation, although the preimmune IgG is not inhibitory. For comparison, theeffect of TC3a alone is shown. To show that the effect of TC3a was spe-cific, the action of the desarginated form of C3a (desArg TC3a) on [Ca2�]i

mobilization was also analyzed. The values represent the average peakfluorescence of all analyzed cells (10–15 cells/experiment; n 3) after10-min incubation with TC3a in the presence or the absence of preimmuneIgG or anti-TC3aR IgG. The asterisk indicates a significant difference (p �0.01) between TC3a- and desArg TC3a-treated cells as well as betweenpreimmune IgG- and anti-TC3aR IgG-treated cells.


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Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a...

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