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The Role of the N-terminal Domain of the Complement Fragment Receptor C5L2 in Ligand Binding

2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês

10.1074/jbc.m609178200

ISSN

1083-351X

Autores

Anne-Marie Scola, Adrian Higginbottom, Lynda J. Partridge, Robert C. Reid, Trent M. Woodruff, Stephen M. Taylor, David P. Fairlie, Peter N. Monk,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10–100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg. C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10–100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg. Complement fragment 5a (C5a) is a 74-residue polypeptide that is a multifunctional proinflammatory mediator that causes leukocyte chemoattraction and degranulation, increases vascular permeability, and stimulates cytokine secretion (1Guo R.F. Ward P.A. Annu. Rev. Immunol. 2005; 23: 821-852Crossref PubMed Scopus (721) Google Scholar). The C-terminal Arg is rapidly cleaved in vivo to form C5a des Arg, a plasma-stable metabolite that has a different spectrum of activities than intact C5a (2Eglite S. Pluss K. Dahinden C.A. J. Immunol. 2000; 165: 2183-2189Crossref PubMed Scopus (53) Google Scholar). The classical receptor for C5a (C5aR) is a member of the G protein-coupled receptor superfamily (3Gerard N.P. Gerard C. Nature. 1991; 349: 614-617Crossref PubMed Scopus (561) Google Scholar, 4Boulay F. Mery L. Tardif M. Brouchon L. Vignais P. Biochemistry. 1991; 30: 2993-2999Crossref PubMed Scopus (192) Google Scholar) and has high affinity for intact C5a but 10–100-fold lower affinity for C5a des Arg (5Cain S.A. Coughlan T. Monk P.N. Biochemistry. 2001; 40: 14047-14052Crossref PubMed Scopus (26) Google Scholar). The receptor N terminus is required for high affinity binding of C5a but not for receptor activation (6Mery L. Boulay F. Eur. J. Haematol. 1993; 51: 282-287Crossref PubMed Scopus (21) Google Scholar, 7DeMartino J.A. Van Riper G. Siciliano S.J. Molineaux C.J. Konteatis Z.D. Rosen H. Springer M.S. J. Biol. Chem. 1994; 269: 14446-14450Abstract Full Text PDF PubMed Google Scholar): a series of acidic and oxygen-sulfated tyrosine residues interact with basic residues in the core of C5a (8Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 9Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Kuhn J. Gerard C. Gerard N.P. Choe H. J. Exp. Med. 2001; 193: 1059-1066Crossref PubMed Scopus (74) Google Scholar). A second distinct binding site is formed by charged residues in the second and third extracellular loops and the external faces of the transmembrane helical bundle and hydrophobic residues in the core of the receptor (10Gerber B.O. Meng E.C. Dotsch V. Baranski T.J. Bourne H.R. J. Biol. Chem. 2001; 276: 3394-3400Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This site is responsible for receptor activation and is the target for agonist and antagonist peptidic mimics of the C terminus of C5a (11Higginbottom A. Cain S.A. Woodruff T.M. Proctor L.M. Madala P.K. Tyndall J.D. Taylor S.M. Fairlie D.P. Monk P.N. J. Biol. Chem. 2005; PubMed Google Scholar). The second C5a receptor to be discovered, C5L2, does not appear to couple to G proteins and appears to have an anti-inflammatory function, balancing the pro-inflammatory role of C5aR (12Gavrilyuk V. Kalinin S. Hilbush B.S. Middlecamp A. McGuire S. Pelligrino D. Weinberg G. Feinstein D.L. J. Neurochem. 2005; 92: 1140-1149Crossref PubMed Scopus (55) Google Scholar, 13Gao H. Neff T.A. Guo R.F. Speyer C.L. Sarma J.V. Tomlins S. Man Y. Riedemann N.C. Hoesel L.M. Younkin E. Zetoune F.S. Ward P.A. FASEB J. 2005; 19: 1003-1005Crossref PubMed Scopus (107) Google Scholar, 14Gerard N.P. Lu B. Liu P. Craig S. Fujiwara Y. Okinaga S. Gerard C. J. Biol. Chem. 2005; 280: 39677-39680Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The mechanism of the anti-inflammatory effect is not known but may be related to the ligand binding properties of C5L2, which has nearly equal affinities for C5a and C5a des Arg. C5L2 has 41% sequence identity with C5aR (15Cain S.A. Monk P.N. J. Biol. Chem. 2002; 277: 7165-7169Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), with a similar array of acidic and tyrosine residues at the N terminus, and many of the charged and hydrophobic residues in the loops and transmembrane regions of C5aR that are involved in the interaction with the C terminus of C5a are also conserved in C5L2.Although recent reports have identified a C5aR agonist peptide as a ligand at C5L2 (16Okinaga S. Slattery D. Humbles A. Zsengeller Z. Morteau O. Kinrade M.B. Brodbeck R.M. Krause J.E. Choe H.R. Gerard N.P. Gerard C. Biochemistry. 2003; 42: 9406-9415Crossref PubMed Scopus (211) Google Scholar, 17Otto M. Hawlisch H. Monk P.N. Muller M. Klos A. Karp C.L. Kohl J. J. Biol. Chem. 2004; 279: 142-151Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the mechanism of ligand binding to C5L2 has not been reported. In this report, we describe a systematic analysis of the role of the N terminus of C5L2 in the interaction with ligands and demonstrate that this domain has a critical role in binding to C5a des Arg but not to C5a.EXPERIMENTAL PROCEDURESCell Culture—RBL-2H3 and Chinese hamster ovary (CHO) 2The abbreviations used are: CHO, Chinese hamster ovary; C5a, complement fragment 5a; C5aR, human complement fragment 5a receptor; Cha, cyclohexylalainine; WT, wild type. cells were routinely cultured in Dulbecco's modified Eagle's medium + 10% (v/v) fetal calf serum supplemented with 400 mg/liter G-418 for transfected cells, at 37 °C, 5% CO2. In experiments where NaClO3 was used to inhibit tyrosine sulfation, cells were grown for 5 days in low sulfate Dulbecco's modified Eagle's medium/F12 (Sigma) supplemented with 10% (v/v) dialyzed fetal calf (Invitrogen) serum and 10 mm NaClO3 (Sigma).Receptor Cloning and Expression—Mouse C5L2 cDNA was kindly provided by Dr. Hui Tian, Amgen Inc. Human C5L2 was cloned as previously described (15Cain S.A. Monk P.N. J. Biol. Chem. 2002; 277: 7165-7169Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). All constructs were cloned into pEE6 (Celltech) and transfected into CHO cells by standard electroporation protocols. After selection in G418, homogenous populations of cells were produced by two rounds of fluorescence-activated cell sorting using rabbit polyclonal anti-sera that recognize the N-terminal sequence of human, rat, or mouse C5L2 on a BD Biosciences Vantage flow cytometer (mouse, human) or cloning by limiting dilution (rat). RBL-2H3 cells expressing mouse C5aR were a generous gift from Dr. Jorg Zwirner (Gottingen); CHO cells expressing human C5aR were made as previously described (18Pease J.E. Barker M.D. Biochem. Mol. Biol. Int. 1993; 31: 719-726PubMed Google Scholar). Rat C5aR, cloned from rat liver using sequence Y09613 and rat C5L2 cDNA, a generous gift from Dr. Vitaliy Gavrilyuk, (University of Illinois), were subcloned into pEE6 for expression in CHO cells. The cDNA for human C5aR with the mutations D15A, D18A, or Y14F in pCDNA3.1 were generous gifts from Dr. Carla de Haas (Utrecht). A monoclonal antibody (M2; Sigma) that recognizes an N-terminal FLAG tag sequence was used to sort the highest 50% of transfected cells in two rounds of fluorescence-activated cell sorting.Production of Peptides, Antibodies, and Recombinant Ligands—Antiserum against a synthetic peptide analog of the N terminus of human C5L2 (1Guo R.F. Ward P.A. Annu. Rev. Immunol. 2005; 23: 821-852Crossref PubMed Scopus (721) Google Scholar, 2Eglite S. Pluss K. Dahinden C.A. J. Immunol. 2000; 165: 2183-2189Crossref PubMed Scopus (53) Google Scholar, 3Gerard N.P. Gerard C. Nature. 1991; 349: 614-617Crossref PubMed Scopus (561) Google Scholar, 4Boulay F. Mery L. Tardif M. Brouchon L. Vignais P. Biochemistry. 1991; 30: 2993-2999Crossref PubMed Scopus (192) Google Scholar, 5Cain S.A. Coughlan T. Monk P.N. Biochemistry. 2001; 40: 14047-14052Crossref PubMed Scopus (26) Google Scholar, 6Mery L. Boulay F. Eur. J. Haematol. 1993; 51: 282-287Crossref PubMed Scopus (21) Google Scholar, 7DeMartino J.A. Van Riper G. Siciliano S.J. Molineaux C.J. Konteatis Z.D. Rosen H. Springer M.S. J. Biol. Chem. 1994; 269: 14446-14450Abstract Full Text PDF PubMed Google Scholar, 8Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 9Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Kuhn J. Gerard C. Gerard N.P. Choe H. J. Exp. Med. 2001; 193: 1059-1066Crossref PubMed Scopus (74) Google Scholar, 10Gerber B.O. Meng E.C. Dotsch V. Baranski T.J. Bourne H.R. J. Biol. Chem. 2001; 276: 3394-3400Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 11Higginbottom A. Cain S.A. Woodruff T.M. Proctor L.M. Madala P.K. Tyndall J.D. Taylor S.M. Fairlie D.P. Monk P.N. J. Biol. Chem. 2005; PubMed Google Scholar, 12Gavrilyuk V. Kalinin S. Hilbush B.S. Middlecamp A. McGuire S. Pelligrino D. Weinberg G. Feinstein D.L. J. Neurochem. 2005; 92: 1140-1149Crossref PubMed Scopus (55) Google Scholar, 13Gao H. Neff T.A. Guo R.F. Speyer C.L. Sarma J.V. Tomlins S. Man Y. Riedemann N.C. Hoesel L.M. Younkin E. Zetoune F.S. Ward P.A. FASEB J. 2005; 19: 1003-1005Crossref PubMed Scopus (107) Google Scholar, 14Gerard N.P. Lu B. Liu P. Craig S. Fujiwara Y. Okinaga S. Gerard C. J. Biol. Chem. 2005; 280: 39677-39680Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 15Cain S.A. Monk P.N. J. Biol. Chem. 2002; 277: 7165-7169Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Okinaga S. Slattery D. Humbles A. Zsengeller Z. Morteau O. Kinrade M.B. Brodbeck R.M. Krause J.E. Choe H.R. Gerard N.P. Gerard C. Biochemistry. 2003; 42: 9406-9415Crossref PubMed Scopus (211) Google Scholar, 17Otto M. Hawlisch H. Monk P.N. Muller M. Klos A. Karp C.L. Kohl J. J. Biol. Chem. 2004; 279: 142-151Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Pease J.E. Barker M.D. Biochem. Mol. Biol. Int. 1993; 31: 719-726PubMed Google Scholar, 19Kalant D. Cain S.A. Maslowska M. Sniderman A.D. Cianflone K. Monk P.N. J. Biol. Chem. 2003; 278: 11123-11129Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 20Lanza T.J. Durette P.L. Rollins T. Siciliano S. Cianciarulo D.N. Kobayashi S.V. Caldwell C.G. Springer M.S. Hagmann W.K. J. Med. Chem. 1992; 35: 252-258Crossref PubMed Scopus (61) Google Scholar, 21de Laszlo S.E. E.E.A. Li B. Ondeyka D. Rivero R. Malkowitz L. Molineaux C. Siciliano S.J. Springer M.S. Greenlee W.J. Mantlo N. Bioorg. Med. Chem. Lett. 1997; 7: 213-218Crossref Scopus (23) Google Scholar, 22Paczkowski N.J. Finch A.M. Whitmore J.B. Short A.J. Wong A.K. Monk P.N. Cain S.A. Fairlie D.P. Taylor S.M. Br. J. Pharmacol. 1999; 128: 1461-1466Crossref PubMed Scopus (80) Google Scholar, 23Wilken H.C. Rogge S. Gotze O. Werfel T. Zwirner J. J. Immunol. Methods. 1999; 226: 139-145Crossref PubMed Scopus (10) Google Scholar, 24Wilken H.C. Gotze O. Werfel T. Zwirner J. Immunol. Lett. 1999; 67: 141-145Crossref PubMed Scopus (33) Google Scholar, 25Moore K.L. J. Biol. Chem. 2003; 278: 24243-24246Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 26Morgan E.L. Ember J.A. Sanderson S.D. Scholz W. Buchner R. Ye R.D. Hugli T.E. J. Immunol. 1993; 151: 377-388PubMed Google Scholar, 27Oppermann M. Raedt U. Hebell T. Schmidt B. Zimmermann B. Gotze O. J. Immunol. 1993; 151: 3785-3794PubMed Google Scholar, 28Crass T. Ames R.S. Sarau H.M. Tornetta M.A. Foley J.J. Kohl J. Klos A. Bautsch W. J. Biol. Chem. 1999; 274: 8367-8370Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 29Sun J. Ember J.A. Chao T.H. Fukuoka Y. Ye R.D. Hugli T.E. Protein Sci. 1999; 8: 2304-2311Crossref PubMed Scopus (21) Google Scholar, 30Postma B. Kleibeuker W. Poppelier M.J. Boonstra M. Van Kessel K.P. Van Strijp J.A. de Haas C.J. J. Biol. Chem. 2005; 280: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 31Mery L. Boulay F. J. Biol. Chem. 1994; 269: 3457-3463Abstract Full Text PDF PubMed Google Scholar, 32Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar) was prepared as described previously (19Kalant D. Cain S.A. Maslowska M. Sniderman A.D. Cianflone K. Monk P.N. J. Biol. Chem. 2003; 278: 11123-11129Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and affinity purified using the synthetic peptide. Compounds 73 and 74 (non-peptidic antagonists) (20Lanza T.J. Durette P.L. Rollins T. Siciliano S. Cianciarulo D.N. Kobayashi S.V. Caldwell C.G. Springer M.S. Hagmann W.K. J. Med. Chem. 1992; 35: 252-258Crossref PubMed Scopus (61) Google Scholar) and 3257 and 3261 (non-peptidic agonists) (21de Laszlo S.E. E.E.A. Li B. Ondeyka D. Rivero R. Malkowitz L. Molineaux C. Siciliano S.J. Springer M.S. Greenlee W.J. Mantlo N. Bioorg. Med. Chem. Lett. 1997; 7: 213-218Crossref Scopus (23) Google Scholar) at the human C5a receptor are described in supplemental material. His6-tagged recombinant ligands and peptides were synthesized as described previously (22Paczkowski N.J. Finch A.M. Whitmore J.B. Short A.J. Wong A.K. Monk P.N. Cain S.A. Fairlie D.P. Taylor S.M. Br. J. Pharmacol. 1999; 128: 1461-1466Crossref PubMed Scopus (80) Google Scholar).Construction of Mutants of Human C5L2 N Terminus—Substitution mutants (E9A, D12A, D15A, D18A, D22A, D25A, Y8F, Y10F, Y13F) were made using Stratagene QuikChange kits according to the manufacturer's instructions and cloned into pEE6 after authentication by DNA sequencing. A chimeric C5L2, with the N terminus of hC5aR, substituting residues 1–32 (hC5L2) with 1–37 (hC5aR), was made by overlap extension using the primers shown in supplemental material. After authentication by DNA sequencing, chimera C5aRNC5L2 was cloned into pEE6 for transfection of CHO cells.His6-C5a/C5a des Arg Binding Assays—The binding of ligand to wild type and mutant C5L2 and C5aR was measured using an indirect immunofluorescence method as previously described (23Wilken H.C. Rogge S. Gotze O. Werfel T. Zwirner J. J. Immunol. Methods. 1999; 226: 139-145Crossref PubMed Scopus (10) Google Scholar, 24Wilken H.C. Gotze O. Werfel T. Zwirner J. Immunol. Lett. 1999; 67: 141-145Crossref PubMed Scopus (33) Google Scholar). Cells transfected with the appropriate receptor (50,000/well of a 96-well microtiter plate) were incubated with the stated concentrations of His6-tagged C5a or C5a des Arg for 30 min at 4 °C in phosphate-buffered saline + 0.1% bovine serum albumin, 0.2% (w/v) NaN3 and then washed twice with cold phosphate-buffered saline to remove unbound ligand. Anti-His6 antibody (mouse IgG; Qiagen) was added (50 μl at 2 μg/ml) and incubated with the cells for 30 min at 4 °C. After a further wash in phosphate-buffered saline, 50 μl of fluorescein isothiocyanate anti-mouse IgG (Sigma) was used to detect bound ligand using a FACSsort flow cytometer (BD Biosciences). Binding was calculated as the percentage of positive cells, i.e. with a fluorescent intensity higher than control cells measured in the absence of ligand. Receptor affinities are reported as EC50 values, calculated by non-linear regression analyses using GraphPad Prism v4.0 (GraphPad Software, San Diego, CA).RESULTSRodent C5L2 Homologs Are Primarily C5a des Arg-binding Proteins—Although human C5L2 has been well characterized, ligand binding by rodent C5L2 has not previously been reported. Rat, mouse, and human C5aR and C5L2 were expressed in CHO cells (except for mouse C5aR, which was expressed in RBL cells), and high expressing cell populations were produced either by cell sorting or by cloning. His6-tagged human, rat, and mouse C5a and C5a des Arg at a high, intermediate, or low dose (5000, 50, 5 nm) determined by the previously reported ligand binding affinities of hC5L2 (15Cain S.A. Monk P.N. J. Biol. Chem. 2002; 277: 7165-7169Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Okinaga S. Slattery D. Humbles A. Zsengeller Z. Morteau O. Kinrade M.B. Brodbeck R.M. Krause J.E. Choe H.R. Gerard N.P. Gerard C. Biochemistry. 2003; 42: 9406-9415Crossref PubMed Scopus (211) Google Scholar) were allowed to bind at 4 °C, followed by extensive washing. Surface-bound ligand was then detected by indirect immunofluorescence using an anti-His6 monoclonal antibody. This method measures only the ligand that remains bound after ∼1.5 h (i.e. that has a slow off-rate) and so C5a des Arg binding to C5aR, for example, appears quite low (Fig. 1) and no binding of human C3a or C3a des Arg to human C5L2 can be detected (data not shown). However, the EC50 values for hC5a binding to both hC5L2 (∼7nm) and hC5aR (∼4nm) (Table 1) are in close agreement with previously published binding affinities obtained using radiolabeled ligands, ∼3nm for both receptors (16Okinaga S. Slattery D. Humbles A. Zsengeller Z. Morteau O. Kinrade M.B. Brodbeck R.M. Krause J.E. Choe H.R. Gerard N.P. Gerard C. Biochemistry. 2003; 42: 9406-9415Crossref PubMed Scopus (211) Google Scholar). Similarly, hC5a des Arg binding to hC5L2 and hC5aR produced EC50 values of ∼36 and ∼527 nm (Table 3), again not dissimilar to affinity values obtained indirectly using radio competition assays, (∼12 and ∼660 nm, respectively) (16Okinaga S. Slattery D. Humbles A. Zsengeller Z. Morteau O. Kinrade M.B. Brodbeck R.M. Krause J.E. Choe H.R. Gerard N.P. Gerard C. Biochemistry. 2003; 42: 9406-9415Crossref PubMed Scopus (211) Google Scholar). Thus, the ligand binding assay employed here is a valid quantitative method for the analysis of receptor affinities, with the advantage that it does not require chemical modification of the ligand and so is used here to facilitate the comparison of ligand binding to receptors from different species.TABLE 1The binding of C5a and C5a des Arg to C5L2 mutated at N-terminal acidic residuesReceptor LigandWTE9AD12AD15AD18AD22AD25AEC50aEC50, concentration (nm) resulting in 50% of the maximum binding.PEC50bpEC50, —log10EC50.ncn, number of separate experiments performed in duplicate.EC50pEC50nEC50pEC50nEC50pEC50nEC50pEC50nEC50pEC50nEC50pEC50nC5a6.928.16 ± 0.05183.108.51 ± 0.08**49.378.03 ± 0.08ns67.108.15 ± 0.09ns43.888.41 ± 0.10*422.97.64 ± 0.08***42.598.59 ± 0.14**4C5a desArg36.07.44 ± 0.071894.57.02 ± 0.12*44036.40 ± 0.13***62526.60 ± 0.10***448.67.31 ± 0.07ns4-<3433.47.48 ± 0.15ns4a EC50, concentration (nm) resulting in 50% of the maximum binding.b pEC50, —log10EC50.c n, number of separate experiments performed in duplicate. Open table in a new tab TABLE 3The binding of C5a and C5a des Arg to C5aR mutated at N-terminal acidic and tyrosyl residuesLigandWTD15AD18AY14FEC50aEC50, concentration (nm) resulting in 50% of the maximum binding.pEC50bpEC50, -log10EC50.ncn, number of separate experiments performed in duplicate.EC50pEC50nEC50pEC50nEC50pEC50nC5a5.528.26 ± 0.05834.17.47 ± 0.14***417.07.71 ± 0.06***841.87.38 ± 0.06***6C5a desArg5276.28 ± 0.14828805.541** ± 0.15443905.36 ± 0.12***8121004.92 ± 0.06***6a EC50, concentration (nm) resulting in 50% of the maximum binding.b pEC50, -log10EC50.c n, number of separate experiments performed in duplicate. Open table in a new tab It can be seen that C5L2 of all three species has an equal or higher affinity (or slower off-rate) for C5a des Arg compared with C5a. For the rodent species in particular, the lower levels of C5a binding suggest that C5L2 is primarily a C5a des Arg-binding protein, with a much lower propensity to bind or retain C5a. Mouse C5L2 is still saturated at 5 nm mC5a des Arg, whereas the binding of 5 nm mC5a is 4000-fold lower than mC5a des Arg (data not shown). This is in contrast to mC5aR, which clearly shows a preference for mC5a over mC5a des Arg (Fig. 1). For rat C5L2, the difference is also marked, with the binding of 5 nm rC5a reduced to almost undetectable levels but 5 nm rC5a des Arg still at near-maximal binding (Fig. 1). The species of C5a/C5a des Arg used also appears to show differences between C5L2 and C5aR. Human C5L2 appears to show promiscuous binding with little preference for same species ligands, whereas human C5aR shows a higher degree of discrimination. Mouse and rat C5L2, in contrast, bind human C5a and C5a des Arg only weakly but show a clear preference for C5a des Arg over C5a (Fig. 1).A Panel of Synthetic Analogs of the C-terminal of C5a Are More Potent Inhibitors at C5aR than at C5L2—For C5aR, the full agonist potential of C5a is vested in only the C-terminal decapeptide of C5a, which binds in the transmembrane binding pocket. We have used a panel of peptidic and non-peptidic analogs of the C5a C terminus to compare the characteristics of the transmembrane binding pockets of C5L2 and C5aR. All the analogs were capable of blocking the binding of 50 nm His6-hC5a to hC5aR at statistically significant levels (Fig. 2A): F-[OP(DCha)WR], YSFKPMPLaR, FKP(DCha)Cha(D-R)-CO2H, FKP(DCha)Cha(D-R)-CONH2, 73, 74, 3261, 3257 (all 100 μm), W-54011 and YFKAChaChaL(D-F)R (both 10 μm because of limited solubility and toxicity, respectively). In contrast, these inhibitors had very weak effects on His6-hC5a binding to hC5L2 (Fig. 2B), and only 3257, 3261, and FKPDChaChaD-R-CONH2 inhibited binding to a significant extent. 3257, 3261, FKPDChaChaD-R-CO2H, and FKPDChaChaD-R-CONH2 could partly inhibit His6-hC5a des Arg binding to hC5L2 (Fig. 2C). The analogs were all ineffective at mC5L2, although only mC5a des Arg binding was measured as mC5a binding levels were too low for accurate assessment of inhibition (Fig. 2D). Thus, despite the conservation of many of the same residues, ligand binding to the transmembrane binding pockets of C5aR and C5L2 is clearly different.FIGURE 2The binding of analogs of the C terminus of C5a to human C5L2 and C5aR. CHO cells transfected with either human C5aR (A) human C5L2 (B, C), or mouse C5L2 (D) were preincubated with high doses (YFKAChaChaLD-FR, W-54011 10 μm, remainder 100 μm) of C5a analogs for 15 min and then incubated for 30 min with 50 nm His6-tagged human C5a (A, B), human C5a des Arg (C), or mouse C5a des Arg (D) and bound ligand quantified by indirect immunofluorescence, expressed as percent of binding to cells in the absence of analogs. The bars are means ± S.E. of four to seven separate experiments performed in duplicate. Significance of difference from 100 was assessed by one sample t test, *, p < 0.5%; **, p < 0.5%; ***, p < 0.05%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)An Antibody Raised against the N Terminus of Human C5L2 Inhibits C5a des Arg, but Not C5a, Binding—To examine the role of the N terminus of C5L2 in ligand binding, a rabbit polyclonal anti-serum, prepared using a synthetic peptide with the N-terminal sequence of human C5L2-(1–32) (19Kalant D. Cain S.A. Maslowska M. Sniderman A.D. Cianflone K. Monk P.N. J. Biol. Chem. 2003; 278: 11123-11129Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), was used. The affinity-purified anti-serum was preincubated at 0.28 mg/ml with hC5L2 before the binding affinity for C5L2 ligands was measured. For hC5L2, the high affinity binding of His6-hC5a was not affected at all by antibody pretreatment (p = 0.62, Student t test) but the affinity for hC5a des Arg was decreased by more than 370-fold (pEC50 values significantly different, p = 0.0006) (Fig. 3. The lack of an effect on hC5a binding suggests that the antibody is blocking binding of C5a des Arg by a specific mechanism, possibly by hindering access to key residues in the N-terminal domain of C5L2.FIGURE 3The effects of an antibody against the N terminus of human C5L2 on ligand binding. CHO cells transfected with human C5L2 were preincubated with phosphate-buffered saline (filled symbols) or 0.28 mg/ml of affinity-purified rabbit polyclonal antibody (open symbols) raised against the N terminus of human C5L2 for 15 min and then incubated for 30 min with His6-tagged human C5a (squares) or C5a des Arg (triangles). Bound ligand was quantified by indirect immunofluorescence and expressed as percent of maximal binding to cells, i.e. in the absence of antibody. The data are means ± S.E. of three separate experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Replacement of the C5L2 N-terminal Domain with C5aR Sequence Does Not Decrease Affinity for C5a des Arg—The inhibition of hC5a des Arg binding by anti-hC5L2 antibody implicates the N terminus in ligand binding. The role of the N-terminal domain was further investigated by making a chimeric form of hC5L2 with the N terminus of hC5aR, substituting residues 1–32 (hC5L2) with 1–37 (hC5aR). After transfection and selection of CHO cells expressing similar levels of receptors, the binding affinity of His6-hC5a and hC5a des Arg was measured (Fig. 4). The affinity of the chimeric receptor, C5aRNC5L2, for hC5a was changed only slightly (<2-fold change relative to both parental receptors). Interestingly, hC5a des Arg binding was also unaffected; the affinity of C5aRNC5L2 was not significantly decreased ( 15-fold). This finding suggests that the N terminus is not the only determinant of the high affinity of hC5L2 for hC5a des Arg.FIGURE 4The binding of C5a and C5a des Arg to human C5L2, C5aR, and an N-terminal chimera. CHO cells transfected with human C5L2, C5aR, and a chimera of human C5L2 with the N-terminal domain of C5aR (C5aRNC5L2) were incubated for 30 min with His6-tagged C5a (A) or C5a des Arg (B) and bound ligand quantified by indirect immunofluorescence, expressed as percent of binding to cells in the absence of analogs. The data are means ± S.E. of three separate experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutation of the C5L2 N Terminus Primarily Affects Binding Affinity for C5a des Arg—The data from the chimeric receptor suggested that common ligand binding elements were present in the N termini of both C5aR and C5L2. To try and identify these elements, we measured the binding affinities of C5L2 mutated at residues analogous to those previously shown to be involved in C5a binding by C5aR: 6 acidic (E9A, D12A, D15A, D18A, D22A, D25A) (Fig. 5, A and B, Table 1) and 3 tyrosine (Y8F, Y10F, Y13F) (Fig. 5, C and D, Table 2) residues in the N terminus of hC5L2. The affinity for hC5a was either unchanged or actually increased in all cases except D22A, where affinity decreased by ∼8-fold. In contrast, the affinity for hC5a des Arg was significantly reduced in several cases: 3

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