Structure-Function Relationships of Human C5a and C5aR Markus S. Huber-Lang, J. Vidya Sarma, Stephanie R. McGuire, Kristina T. Lu, Vaishalee A. Padgaonkar, Ellen M. This information is current as Younkin, Ren Feng Guo, Christian H. Weber, Erik R. of September 23, 2021. Zuiderweg, Firas S. Zetoune and Peter A. Ward J Immunol 2003; 170:6115-6124; ; doi: 10.4049/jimmunol.170.12.6115

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Structure-Function Relationships of Human C5a and C5aR1

Markus S. Huber-Lang,* J. Vidya Sarma,* Stephanie R. McGuire,* Kristina T. Lu,* Vaishalee A. Padgaonkar,* Ellen M. Younkin,* Ren Feng Guo,* Christian H. Weber,* Erik R. Zuiderweg,† Firas S. Zetoune,* and Peter A. Ward2*

Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four ␣ helices of C5a, we have defined the ability of these peptides to reduce binding of 125I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H2O2 production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a. The Journal of Immunology, 2003, 170: 6115–6124. Downloaded from

uman C5a (C5a) is a 74-aa glycoprotein generated upon around the fifth transmembrane domain (“effector site”), leading activation of the via the classical, via G activation to signal transduction (21). In recent stud- H alternative, or lectin-binding pathways (1). In concert ies, a genetic screen of C5aR mutants has identified in C5aR two with , and the membrane attack complex (C5b-9), C5a distinct clusters of residues that possibly serve as a C5a-activated plays a central role in host defenses. Excessive complement acti- “molecular switch” (22). One cluster, located at the extracellular vation leading to elevated plasma levels of C5a is known to be face of the receptor (binding area), may be required for interaction http://www.jimmunol.org/ associated with many clinical conditions, including sepsis (2), with the C5a molecule and the second cluster, at the core of the adult respiratory distress syndrome (3), rheumatoid arthritis (4), transmembranous helix bundle, may transmit the signal (via con- Alzheimer’s disease (5), and ischemic heart disease (6), to name formational changes) from the extracellular C5a ligand to the G just a few conditions. Biochemical and pharmacological studies protein (22–24). indicate that both C3a and C5a bind and interact with receptors For the human C3aR, direct binding and calcium mo- that belong to the G protein-coupled superfamily of -like bilization assays have shown that the large second extracellular receptors (7–10). The C5aR (CD88) is expressed widely on in- loop (between the fourth and fifth transmembrane domain) is nec-

flammatory (9–12) and noninflammatory cells (13–18). For C5a- essary not only for high-affinity C3a binding but also for the sub- by guest on September 23, 2021 C5aR interaction, multiple discontinuous residues in two major sequent functional responses (25). In contrast, for the C5aR and structural elements within the C5a molecule have been reported to IL-8 receptors, the N terminus and the third extracellular loop A and B be required, as identified by nuclear magnetic resonance (NMR) have been suggested to contain structures critical for binding (26) structure analysis and site-directed mutagenesis experiments (19, but redundant for activation. With regard to the ligands, all binding 20). The first site of interaction is the highly ordered disulfide energy is contributed by the C terminus of C3a, in contrast to C5a bridge-stabilized core of C5a (aa 1–63) with its four ␣ helices where the central portion contributes a considerable amount of juxtaposed in an antiparallel topology and connected by three pep- receptor interaction energy (27), suggesting, that different regions tide loops. The second area of C5a implicated in interaction with of the C5a molecule have to act in concert to achieve full potency. C5aR is the relatively disordered and flexible carboxyl-terminal Whereas many studies have focused on the C terminus of C5a tail of C5a (aa 64–74) (20). To understand the interplay between (which interacts with the “effector site” of the C5aR), little is C5a and C5aR that trigger signal pathways, a “two-site binding” known about different regions within the middle of C5a (and their model of C5a-C5aR interaction has been proposed (21). Whereas interactions with C5aR), especially the inter-␣ helical peptide the disulfide-linked core region of C5a may be recognized and loops. Therefore, in the present study the structure-function rela- have binding interactions with both the N-terminal segment as well tionships of different selected peptide regions of C5a were inves- as the third extracellular loop of C5aR (“recognition site”), the tigated. Loop peptide homologues, which are chemically con- C-terminal region of C5a may fit into a binding pocket formed served within the family of C5a species and are structurally different from the corresponding residues in the family of C3a species (20), were investigated as candidates for additional inter- *Department of Pathology, University of Michigan Medical School, Ann Arbor, MI action of C5a with its receptor. Based on present data, a “three- 48109; and †University of Michigan, Biophysics Research Division, Ann Arbor, MI site” interaction of C5a with C5aR is proposed. 48109 Received for publication November 20, 2002. Accepted for publication April 8, 2003. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Reagents charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Human rC5a was purchased from Sigma-Aldrich (St. Louis, MO). All ma- 1 This study was supported by National Institutes of Health Grants GM 61656 (to terials were obtained from Sigma-Aldrich unless otherwise indicated. P.A.W.) and GM 29507 (to P.A.W.). Selection and preparation of peptides of human C5a 2 Address correspondence and reprint requests to Dr. Peter A. Ward, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Ar- Based on structure-function studies (19, 20) of human complement C5a bor, MI 48109-0602. E-mail address: [email protected] (C5a), different regions of the molecule were chosen and synthesized for

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 6116 STRUCTURE-FUNCTION OF C5a AND C5aR evaluation of their ability to interact with the C5aR and affect the biological 37°C. The number of cells that migrated through the polycarbonate mem- responses of neutrophils. The amino-terminal “A” region with the sequence brane to the lower surface was determined by cytofluorometry (Cytofluor MLQKKIEEIAAKYKHSVVKK (corresponding to aa residues 1–20), the II; PerSeptive Biosystems, Framingham, MA). For each measurement, middle “M” region CCYDGASVNNDETCEQRAAR (corresponding to aa samples were done at least in quadruplicate. residues 21–40), and the carboxyl-terminal “C” region CVVASQLRAN ISHKDMQLGR (corresponding to aa residues 55–74) were selected as Generation of H2O2 by neutrophils peptides of C5a. A scrambled version of the C peptide was also synthesized H O production was determined by an established assay (29). Briefly, (C scrambled: CRVGVLAQSMQDLKRHASIN). Additionally, based on 2 2 isolated neutrophils (2 ϫ 106 the proposed three-dimensional (3D) structure (20), smaller peptide re- cells/ml) were pretreated for1hat37°C with either C5a (10 nM) or the helical peptide regions (described above) A, M, gions, representing the first, second, and third inter-␣ helical loop regions C (each at 1–1000 nM), or the loop peptides D ,D, and D (each at of C5a molecule were synthesized as follows: D (KYKHSVVKK, aa res- 1 2 3 1 10–1000 nM), or HBSS (control) alone in the presence of 1 mM sodium idues 12–20), D (VNNDET, aa residues 28–33), and D (AARISLGPR, 2 3 azide (to prevent endogenous catalases from destroying H O ). To stimu- aa residues 38–46) (Fig. 1A). In addition, scrambled versions of D peptide 2 2 2 late neutrophils, PMA (25 ng/ml) was added at the end of the preincubation were also synthesized (D2-scrambled I: TENFNV; D2-scrambled II: EN- TNDV). These peptides were synthesized and purified by HPLC (Research and the cell suspensions were then incubated at 37°C for 15 min. The Genetics, Huntsville, AL and Open Biosystems, Huntsville, AL). reaction was then terminated by addition of 0.1 ml of trichloroacetic acid (50% w/v). The samples were centrifuged for 10 min at 500 ϫ g and Isolation of human neutrophils ferrous ammonium sulfate and potassium thiocyanate were added to the supernatant fluid at final concentrations of 1.5 mM and 0.25 M, respec- Whole blood from healthy donors was drawn into syringes containing 0.1 tively. The absorbance of the ferrithiocyanate complex was measured at ml/ml blood of anticoagulant ACD (Baxter, Health Care, Deerfield, IL). 480 nm and compared with a standard curve generated from dilutions of

Neutrophils were isolated using Ficoll-Paque (Pharmacia Biotech, Upp- reference solutions of H2O2.

sala, Sweden) gradient centrifugation (1500 rpm, room temperature, 30 Downloaded from min). The cell pellet was resuspended in dextran (final concentration, 1%) Analysis of the molecular structure and determination of followed by a 45-min sedimentation step of erythrocytes. Supernatant flu- possible interaction sites of C5a and C5aR ids containing neutrophils were centrifuged (1500 rpm, room temperature, 5 min) and residual RBCs were removed by a hypotonic lysis step. After To analyze the molecular structures of C5a and C5aR and to localize pos- washing, neutrophils were resuspended in assay buffer and evaluated in sible C5a-C5aR interaction sites, the published sequences of C5a and C5aR binding studies, chemotaxis assays, and oxidative burst measurements. were molecular graphics in conjunction with examined using the PROSITE database (30) and PHD coil prediction (31), and based on our current in 125 I-C5a binding studies vitro data a three-site binding model was designed. http://www.jimmunol.org/ For binding studies, commercially available recombinant human C5a (C5a) Statistical analyses was labeled with 125I using the chloramine-T-based protocol (28), which allows gentle oxidation and preserves the chemotactic activity of C5a for All values were expressed as mean Ϯ SEM. Data sets of binding, chemo- neutrophils (data not shown). To avoid receptor internalization, all of the taxis, and H2O2 assays were analyzed with one-way ANOVA; differences following steps were performed at 4°C. To block nonspecific surface bind- in the mean values among experimental groups were then compared using ing sites, isolated neutrophils were incubated for1hinbinding buffer the Tukey multiple comparison test. Results were considered statistically (HBSS, without Ca2ϩ, containing 1% BSA). After a gentle washing step, significant where p Ͻ 0.05. neutrophils (2 ϫ 106 cells) were incubated in binding buffer (HBSS, with Ca2ϩ, containing 0.1% BSA) in 1.5-ml microcentrifuge tubes (in a final Results

125 by guest on September 23, 2021 volume of 200 ␮l) with 100 pM I-C5a (sp. act., 33.24 ␮Ci/␮g) in the C5a peptide regions selected for study absence or presence of increasing amounts of unlabeled C5a (ranging from 10Ϫ11 to 10Ϫ6 M). After an incubation interval of 20 min, cell suspensions Based on the sequence and the NMR structure of C5a, different were layered over 20% sucrose and sedimented by centrifugation at regions of C5a were chosen for synthesis of peptides. For reference 11,000 ϫ g (Beckman Microfuge B; Beckman Coulter, Palo Alto, CA) for Ϫ purposes, Fig. 1A depicts the NMR structure indicating positions 2 min. The tubes were then frozen at 80°C and the tips, containing the ␣ cell pellet, were cut off to determine the cell-bound 125I-C5a using a of the designated inter- -helical loop peptides (D1,D2,D3). This gamma counter (1261 Multigamma; Wallac, Gaithersburg, MD). Binding figure was generated using MOLSCRIPT (32) and POV-Ray. For affinities (Kd values) of C5a were calculated in the conventional manner reference purposes, the structure of C5a and its proposed interac- (9). The saturation binding curve was generated using increasing amounts tions with C5aR (shown by the red shaded regions) are shown in of 125I-labeled C5a (1–20 nM) in the absence or presence of 100-fold excess of unlabeled C5a and analyzed by nonlinear regression analysis Fig. 1B. using the GraphPad Prism program (GraphPad Software, San Diego, CA). 125 Interference of the different synthetic peptides (1–1000 nM) with C5a bind- Antagonization of I-C5a binding to neutrophils by C5a ing was determined in the absence or presence of 10 nM 125I-labeled C5a peptides using 2 ϫ 106 neutrophils in 200 ␮l of binding buffer. These studies were undertaken to assess the binding of 125I-C5a to Chemotaxis assay blood neutrophils in the presence or absence of the different se- lected peptide regions of C5a. Using 100 pM 125I-C5a, competi- Following neutrophil isolation, cells were fluorescein-labeled with 2Ј,7Ј- bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethyl ester) tion experiments with unlabeled C5a revealed a Kd value of 1 nM (Molecular Probes, Eugene, OR) for 30 min at 37°C. After a washing step, (data not shown), which is consistent with published data (10). The labeled neutrophils (5 ϫ 106 cells/ml) were loaded into the upper chamber saturation binding curve indicating a plateau of binding beyond 10 of a 96-well mini chamber (NeuroProbe, Gaithersburg, MD), separated by nM C5a is shown in the inset of Fig. 2A. Six different peptides a polycarbonate filter with a porosity of 3 ␮m (NeuroProbe). The lower chambers were loaded with different concentrations of C5a or fMLP in the termed A, M, and C (aa residues 1–20, 21–40, and 55–74, respec- absence or presence of different concentrations of synthetic peptides of C5a tively), and loop peptides D1,D2,and D3 (described in Fig. 1) were (ranging from 10 to 1000 nM). Cells were then incubated for 30 min at evaluated for their ability to inhibit the binding of 10 nM 125I-C5a

FIGURE 1. A, Ribbon diagram of the human C5a molecule with the ␣ helices 1–4 (gray bands) and the less well-defined interconnecting interhelical loop regions (narrow regions). According to the 3D structure of human C5a, the selected inter-␣ helical regions are surface residues of interest for possible

C5a-C5aR interaction (D1 representing acid residues 12–20, D2 acid residues 28–33, and D3 acid residues 38–46, respectively). The core is stabilized by disulfide linkages among residues 21–47, 22–54, and 34–55 (yellow). B, Model for the interaction of C5a with C5aR. The seven transmembrane helices (cylinders 1–7) of C5aR contain different charged loop regions; especially the amino-terminal and extracellular loop II with possible interaction sites for the D1 (acid residues 12–20) and D2 (acid residues 28–33) region of C5a. Electrostatic interactions with a possible generation of salt bridges (darker gray areas) in extracellular loops of C5aR) are shown based on computer-assisted structure analysis of the molecule (models adapted from Refs. 20 and 47). The Journal of Immunology 6117 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

Figure 1. 6118 STRUCTURE-FUNCTION OF C5a AND C5aR

motactic responses were assessed using 10 nM C5a (this concen- tration being associated with peak chemotactic response (Fig. 3A, left inset, arrow)). C5a was added to the lower compartments of chemotactic chambers in the presence or absence of the six dif-

ferent peptides, A, M, C (Fig. 3A) and D1,D2, and D3 (Fig. 3B). Using concentrations ranging from 10 to 1000 nM, all peptides failed to induce migrational responses of neutrophils in the ab- sence of C5a (Fig. 3, all open bars representing peptides at 1 ␮M). The dose responses for A, M, and C peptide interference with chemotactic responses to C5a are shown in the right inset for Fig. 3A. In the presence of 10 nM C5a, 1 ␮M peptide M or C (repre- senting residues 21–40 and 55–74, respectively) resulted in a sig- nificant reduction (Ͼ60%) in the chemotactic responses. In sepa- rate and independent experiments, the C peptide (1 ␮M) reduced the neutrophil chemotactic response to C5a by 87%, whereas the scrambled C peptide (1 ␮m) reduced the response by only 8.5% (Table I). The N-terminal helix peptide A failed to significantly alter C5a-induced migration of neutrophils, albeit interference with

C5a binding to neutrophils was found with the same peptide (Fig. Downloaded from 2A), indicating some differences in binding ability and subsequent

effects on functional responses to C5a. Whereas loop peptide D3 did not significantly alter chemotactic responsiveness of neutro- phils to C5a, significant reductions (Ͼ60%) by synthetic loop pep-

tide D2 occurred, while nearly total inhibition by peptide D1 was

found (Fig. 3B). D3 peptide failed to show any inhibitory effect on http://www.jimmunol.org/ the chemotactic response to C5a, consistent with the relatively poor reduction in binding of 125I-C5a (Fig. 2B). In contrast to the

inhibitory effects of D2 peptide on chemotactic responses to C5a (85% reduction, Fig. 3B and Table I), the scrambled D2 peptides (D2-scrambled I and D2-scrambled II) had no effects on the che- motactic responsiveness of neutrophils to C5a (Ͻ7% reductions, Table I). Complete dose responses for the inhibitory activity of FIGURE 2. A, Interference of C5a peptides with human 125I-C5a bind- each D peptide, normalized to the 10 nM C5a response, are shown by guest on September 23, 2021 ing to neutrophils. Different helical peptides, labeled A (representing res- in the right-hand inset of Fig. 3B. These data suggest that peptides idues 1–20), M (residues 21–40), and C (residues 55–74) (each at 1 ␮M) from the middle (M) and carboxyl (C)-terminal regions as well as 125 were used to antagonize the saturated binding of I-C5a (10 nM) to neu- from the loop regions (D1 and D2, but not D3) have the ability to trophils. Inset, Saturation binding curve of C5a (F ϭ 125I-C5a, E ϭ 125I- compete functionally with intact C5a, suggesting multiple sites for C5a in the presence of 100-fold excess of unlabeled C5a). B, Antagoniza- the binding of C5a to C5aR. tion of three small loop peptides D1 (representing residues 12–20), D2 (residues 28–33), and D (residues 38–46) with C5a (none) binding on 3 Specificity of C5a peptides for interference with chemotaxis of human neutrophils. For each data point, at least six independent experi- ments were performed. human neutrophils to C5a but not to fMLP As shown in Figs. 4A and 5A, the dose-dependent chemotactic response of neutrophils to 10 nM C5a was examined in the absence and presence of A, M, and C peptides as well as D ,D, and D to human neutrophils. Except for loop peptide D , all peptides 1 2 3 3 ␮ (each at 1 ␮M) significantly reduced binding of C5a by Ͼ50% peptides (all at 1 M). As expected, both the helical A peptide and the loop D peptide failed to significantly alter the dose-dependent (Fig. 2). Lower concentrations of peptides (1–100 nM) revealed a 3 dose dependency, with no significant alteration of C5a binding C5a-induced chemotactic response of neutrophils over a wide dose when 1–10 nM of peptides were used and Ͼ25% reduction in the range of C5a (0.1 pM to 100 nM of C5a). In contrast, the presence of M and C peptides as well as the D1 and D2 peptides significantly presence of 100 nM for all peptides, exclusive of peptide D3, which again failed to alter binding (data not shown). The pattern of suppressed the C5a-induced dose-dependent chemotactic re- the reduced binding response suggests additional important and, as sponses (Figs. 4A and 5A). Companion experiments were con- ␮ yet unknown, binding sites present in the middle region of C5a ducted to see whether any of the six peptides (all at 1 M) would represented by M peptide and in the interhelical loop peptides affect the chemotactic responses of human neutrophils to a variety of concentrations of fMLP, another potent chemotactic agonist for represented by D1 and D2 in addition to the already designated binding sites in the N- and C-terminal regions of C5a (19–21). As human neutrophils. As shown in Figs. 4B and 5B, none of the would be expected, intact C5a had the most competitive activity in peptides affected the chemotactic responses of human neutrophils terms of interfering with binding of 125I-C5a to human neutrophils. to fMLP. These results demonstrate the specificity of these pep- tides to C5a-induced responses of human neutrophils. C5a peptides reduce C5a-induced chemotactic activity of human

neutrophils Ability of C5a and C5a peptides to inhibit the H2O2 responses Fig. 3 shows the ability of the different synthetic C5a peptides of PMA-stimulated neutrophils (each at 1 ␮M) to reduce chemotactic responses of neutrophils to We have recently shown that in vitro exposure of human neutro- C5a. Human neutrophils were isolated from whole blood and che- phils to human C5a in a dose- and time-dependent manner reduces The Journal of Immunology 6119 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. A, Chemotactic response of neutrophils to C5a (left inset) and C5a (at 10 nM) in the presence or absence of helical peptides (A, M, and C, each at 1 ␮M). Right upper inset, Dose-response inhibition of A, M, and C peptides in chemotactic responses of neutrophils to 10 nM C5a. B, Ability of ␮ different C5a loop peptides (D1,D2, and D3, each at 1 M) to block chemotactic activity of human neutrophils to the whole C5a molecule. Inset, Dose dependency of the peptide-induced interference with chemotaxis of neutrophils to 10 nM C5a. For each experimental end point, n ϭ 6–12. ctrl, Control. 6120 STRUCTURE-FUNCTION OF C5a AND C5aR

a Table I. Ability of C5a peptides to reduce chemotactic and H2O2 responses of neutrophils

Chemotaxis in the Presence H2O2 Response after PMA Incubation with of C5a (10 nM) (25 ng/ml) Stimulation Peptides (1 ␮M) Amino Acid Sequence (% response to C5a) (% response to PMA)

None Not applicable 100.0 Ϯ 2.4 100.0 Ϯ 0.1 C peptide CVVASQLRANISHKDMQLGR 13.2 Ϯ 3.4 15.4 Ϯ 0.4 C scrambled CRVGVLAQSMQDLKRHASIN 91.5 Ϯ 3.4 66.8 Ϯ 0.6 Ϯ Ϯ D2 peptide VNNDET 15.2 2.2 21.5 0.4 Ϯ Ϯ D2 scrambled I TENDNV 93.2 7.0 98.5 0.4 Ϯ Ϯ D2 scrambled II ENTNDV 107 10.1 84.3 0.2

a Chemotaxis and H2O2 assays were performed essentially as described in Materials and Methods. The data are represen- tative of three or more separate and independent experiments performed in triplicate for each condition.

the ability of human neutrophils to generate H2O2 following stim- (Fig. 6), which has recently been shown to be caused by a C5a- ulation with PMA (2, 33). Human neutrophils in suspension were induced inhibition of the NADPH oxidase assembly (33). Pre-ex- incubated for1hat37°C in the presence or absence of peptides A, posure of neutrophils to 1 ␮M of peptides A, M, or C significantly

M,andCorD1,D2, and D3 (in doses ranging from 1 to 1000 nM). reduced PMA-induced generation of H2O2 (Fig. 6A). In separate

As indicated, cells were then exposed to PMA (25 ng/ml) and the and independent experiments, pre-exposure of neutrophils to 1 ␮M Downloaded from

H2O2 response was assessed as described elsewhere (29). The native C peptide reduced the H2O2 response by 85%, whereas the PMA concentration chosen was according to our recently pub- scrambled C peptide (1 ␮M) reduced the response by only 35% lished study, in which the 25-ng/ml dose of PMA exhibited a half- (Table I). Dose responses for all peptides are shown in the insets maximal oxidative burst response of neutrophils (33). As shown in in Fig. 6. Pre-exposure of neutrophils with 1 ␮M of loop peptides

Fig. 6, in the absence of PMA, C5a and the six different peptides D1 or D2 resulted in nearly total abolition of the H2O2 response to

failed to induce significant H2O2 responses (gray bars). The scram- PMA, whereas exposure to the D3 peptide did not significantly http://www.jimmunol.org/ bled C and D2 peptides by themselves also failed to induce H2O2 alter the H2O2 response to PMA (Fig. 6B). In separate and inde- ␮ responses (data not shown). When neutrophils were incubated for pendent experiments, 1 MD2 peptide reduced the H2O2 response ␮ 1hat37°C in buffer and then exposed to PMA in the absence of by 78.5%, while 1 M scrambled D2I and D2II peptides reduced peptides (none), robust H2O2 responses were found (Fig. 6). In the H2O2 responses by only 1.5% and 15.7%, respectively, (Table both sets of experiments, pre-exposure of neutrophils to 10 nM I). These data are consistent with the order of effects of pep-

C5afor1hat37°C almost totally abolished the H2O2 response tides described in Figs. 2–4, as demonstrated by their ability to by guest on September 23, 2021

FIGURE 4. A, Dose dependency of the chemotactic responses of neu- trophils to C5a, ranging from 0.1 pM to 100 nM, or B, fMLP, ranging from FIGURE 5. A, Dose dependency of the chemotactic responses of neu- ␮ 0.1 nM to 1 M in the presence or absence of peptides A, M, and C (each trophils to C5a or B, fMLP in the presence or absence of peptides D1,D2, ␮ ␮ ϭ at 1 M). Number of experiments, 6–10. ctrl, Control. and D3 (each at 1 M). For each end point, n 6–10. ctrl, Control. The Journal of Immunology 6121 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

␮ FIGURE 6. A, Blockade of H2O2 responses in PMA-stimulated neutrophils by C5a and different helical peptides A, M, and C (each at 1 M), or B, and ␮ loop peptides D1,D2, and D3, each at 1 M. Upper inset, Dose responses for A, M, and C peptides in suppressing PMA (25 ng/ml)-induced H2O2 production in neutrophils. Lower inset, Dose-response inhibition for D1,D2, and D3 peptides in PMA-induced H2O2 responses of neutrophils. Number of each experiment: n ϭ 6–10. ctrl, Control. 6122 STRUCTURE-FUNCTION OF C5a AND C5aR interfere with C5a binding to human neutrophils and to inhibit 45) and no functional activity (39, 44) was demonstrated, in con- C5a-induced chemotactic responses. firmation of our findings. In the current studies, neutrophils were exposed to C5a, result-

ing in a defective H2O2 response of stimulated neutrophils. When Discussion human neutrophils were first exposed to the various peptides rep-

The complement system is a main buttress of the host defense resenting regions of C5a, all designated peptides except of D3 were system. The complement activation product, C5a, is known to be capable of profoundly compromising H2O2 responses in a dose- a potent mediator of the -phagocytic cell system and dependent manner (Fig. 5). The D1, M, and D2 peptides, but not binds to the seven-transmembrane spanning receptor, C5aR (10). the scrambled D2 peptides, were potent molecules for inhibiting The parts of the C5a molecule mainly involved in binding to C5aR H2O2 generation, suggesting that important biological interaction and in subsequent biological responses are still matters of debate sites may exist in the middle region of C5a. Mutations in the D1 (34–36). This is in striking contrast to C3a in which the C-terminal region ((A-19, A 20)-rhC5a), but not in the D3 region, have been peptide (residues 69–77) contains virtually all of the biological shown to influence myeloperoxidase release from human neutro- function of intact C3a (37), while C-terminal peptides of C5a con- phils (45). In addition, the amino-terminal peptides 1–17 of por- tain some biological activity (38–40), albeit this is extremely lim- cine C5ades arg have been reported to cause neutrophil degranula- ited. Only at very high concentrations do these peptides show weak tion in guinea pigs and to contribute to the biological potency of agonist activity when compared with intact C5a. This is consistent the intact molecule (43). Other studies, which have investigated with the concept that interaction of C5a with C5aR involves sev- enzyme release from human neutrophils in the presence of cy- eral discontinuous regions of C5a and C5aR (22–24, 32, 41–50). tochalasin B, indicated an effector site in the C-terminal decapep- Downloaded from

Site-directed mutagenesis studies of C5a have suggested that re- tide C5a65–74, with residues 65–69 contributing to biological ac- gions in the middle of the C5a molecule as well as in the C and N tivity (41, 53). This is consistent with our findings suggesting the terminus ends facilitate C5a binding to C5aR on neutrophil mem- biological efficacy of the C peptide region of C5a, in addition to 125 branes (19). Our findings of substantial inhibition of I-C5a bind- the central (middle) region and D1 loop peptide region. ing to intact human neutrophils in the presence of synthetic pep- Almost the same pattern of sites in the C5a molecule interactive

tides representing the amino-terminal, middle, and carboxyl- with C5aR could be identified in chemotaxis assays using different http://www.jimmunol.org/ terminal regions of C5a implicate an involvement of all three peptides in the presence of C5a. Neutrophil exposure to these pep- regions in binding of C5a to C5aR (Fig. 2). Although many studies tides did not affect the chemotactic responses of neutrophils to have focused on the amino- or C-terminal regions, little is known fMLP (Figs. 3 and 4), suggesting specificity of the peptides. Sim- about the binding properties of the middle peptide region of C5a or ilarly, the scrambled peptides had no effects on chemotactic and the loop regions of C5a. In one report, a disulfide-linked core pep- H2O2 responses of neutrophils when compared with the non- tide including residues 20–37 obtained by tryptic digestion of scrambled C and D2 peptides. Mutation of the N terminus of C5a C5adesarg was shown to exhibit weak competitive binding of C5a did not alter chemotactic responses to C5a, whereas chemotactic to C5aR (47), which would support our findings. responses of neutrophils to R-74, G-73, L-72, K-68, or R-40-sub- However, not only linear protein segments but also posttransla- stituted mutants of C5a were significantly reduced (19). Addition- by guest on September 23, 2021 tional changes (51) and assembled topographic sites (i.e., arrays of ally, substitution in C5a at A-26 appeared from NMR studies to amino acids juxtaposed by the 3D folding of the helical region of reduce chemotactic potency of neutrophils by carrying a long-dis-

C5a) are possible targets for binding interactions between C5a and tance conformational change in D1 around H-15, again suggesting C5aR (51, 52). Therefore, based on 3D NMR structure studies, discontinuous central regions and the C terminus to be required to which showed closely conserved internal residues of the C3a and achieve full biological response (19, 41). C5a molecules but completely different surface residues (43, 44), In an attempt to localize sites within C5aR (9, 10) that could we chose different inter-␣ helical loop peptide regions containing interact with the C5a molecule, the secondary structure and po- residues of interest (K-14, H-15, V-17, K-19, E-32, T-33, A-39, tential interactions of C5a and C5aR were evaluated using the R-40, and L-43) as candidates for additional interactions of C5a PROSITE database (30) and PHD coil analysis (31). Based on our with C5aR (20) (Fig. 1). In competitive binding assays, peptides in vitro data, we propose a three-site binding model (Fig. 1B). ␣ representing inter- helical loop peptides (D1 and D2) significantly There is much evidence that a first binding site is located between reduced C5a binding, but blocking activity was not found with the the negatively charged amino residues in region 21–30 of the loop D3 peptide (Figs. 1 and 2). The efficacy of the D1 peptide is C5aR (21, 34, 54) and the four positively charged K-12/14/19/20 supported by other data indicating that a double replacement of residues within the D1 region. The negatively charged N-terminal K-19 and K-20 in this C5a region reduced binding of C5a to C5aR residues 10–18 of C5aR were reported not to be directly involved (45). Further evidence for a possible receptor-interaction site in the in binding of C5a but it was suggested that they interact with the

C5a peptide loop D1 has been suggested by a greatly increased positively charged and hydrophobic stretch of residues within ex- affinity of a conformationally biased C-terminal decapeptide when tracellular C5aR loop II, which is close to the fifth transmembrane linked to C5a12–20 (39). Loop D2, which represents a small peptide helix, presumably forming a stable C5a binding pocket (34). On region within the M peptide of C5a, revealed equal effects on the other end of the extracellular C5aR loop II (close to the fourth blocking the binding of C5a to C5aR in contrast to the M peptide, transmembrane helix), there is an additional positively charged suggesting that the hexapeptide D2 represents a receptor interac- region that could interact with the negatively charged D2 region of tion domain of the middle region. In support of this conclusion, C5a which therefore might be considered as a second, newly rec-

C5a20–37 residues that contain the D2 region have been shown to ognized binding site. The disulfide bond between C-109 in the represent an important receptor recognition domain (47). Incon- extracellular loop I and C-189 in the extracellular loop II (34) sistent results have been reported regarding the interaction of the could stabilize this proposed second binding site. There is also

C5a peptide loop D3 (including residues R-40 and R-46) with fairly good evidence for the existence of a third C5a-C5aR inter- C5aR. Although substitution of R-40 by G changed C5a binding action site (21, 34, 35, 54, 55) in which R-206 in the fifth trans- behavior to neutrophil membranes (19), no participation of this membrane domain has been suggested to play an important role in loop inclusive of R-40 and P-45 in receptor binding was found (38, binding the C terminus by acting as a counterion for R-74 of C5a The Journal of Immunology 6123

(35). A recent report has suggested in neutrophils that further C- 18. Fayyazi, A., O. Scheel, T. Werfel, S. Schweyer, M. Oppermann, O. Gotze, terminal interactions also may occur between K-68 of C5a and H. J. Radzun, and J. Zwirner. 2000. The is expressed in normal renal proximal tubular but not in normal pulmonary or hepatic epithelial cells. Immu- E-199 of the C5aR, close to the fifth transmembrane helix (56). nology 99:38. Another study has shown that substitution of H-67 by F resulted in 19. Mollison, K. W., W. Mandecki, E. R. Zuiderweg, L. Fayer, T. A. Fey, R. A. Krause, R. G. Conway, L. Miller, R. P. Edalji, M. A. Shallcross, et al. 1989. a significant increase in potency of C5a, suggesting an advantage Identification of receptor-binding residues in the inflammatory complement pro- in receptor binding by adding more hydrophobic residues to the tein C5a by site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 86:292. ligand (41), facilitating interactions between the C5a C terminus 20. Zuiderweg, E. R., D. G. Nettesheim, K. W. Mollison, and G. W. Carter. 1989. Tertiary structure of human complement component C5a in solution from nuclear and transmembrane helices of C5aR (21). Furthermore, in com- magnetic resonance data. Biochemistry 28:172. parison to C5aR, the recently published C5L2 (57) 21. Siciliano, S. J., T. E. Rollins, J. DeMartino, Z. Konteatis, L. Malkowitz, (which binds C5a with high affinity but couples poorly to G pro- G. Van Riper, S. Bondy, H. Rosen, and M. S. Springer. 1994. Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein-coupled tein-mediated signaling pathways) exhibit almost identical electri- receptors. Proc. Natl. Acad. Sci. USA 91:1214. cal charge patterns of the three above suggested interaction sites 22. Gerber, B. O., E. C. Meng, V. Do¨tsch, and T. J. Baranski. 2001. An activation (Fig. 1B), even if there is only a 35% amino acid identity switch in the ligand binding pocket of the C5a receptor. J. Biol. Chem. 276:3394. 23. Baranski, T. J., P. Herzmark, O. Lichtarge, B. O. Gerber, J. Trueheart, with C5aR. E. C. Meng, T. Iiri, S. P. Sheikh, and H. R. Bourne. 1999. C5a receptor activation: These data imply that within the C5a molecule at least three genetic identification of critical residues in four transmembrane helices. J. Biol. different discontinuous regions exist and interact with C5aR and Chem. 274:1575. 24. Geva, A., T. B. Lassere, O. Lichtarge, S. K. Pollitt, and T. J. Baranski. 2000. suggest possible targets for the development of effective C5aR Genetic mapping of the human C5a receptor. J. Biol. Chem. 275:35393. antagonists for intervention in the inflammatory response. 25. Chao, T. H., J. A. Ember, M. Wang, Y. Bayon, T. E. Hugli, and R. D. Ye. 1999.

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