1

Europium-labeled synthetic C3a protein as a novel fluorescent probe for

human complement C3a receptor

Aline Dantas de Araujo,†, ‡# Chongyang Wu,†, ‡# Kai-Chen Wu,†, ‡, § Robert C. Reid,†, ‡, § Thomas

Durek,† Junxian Lim,†, ‡, §* David P. Fairlie†, ‡, §,*

†Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia

‡Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072,

Australia §Centre for Inflammation Disease Research,

The University of Queensland, Brisbane, QLD 4072, Australia

#Joint first authors

*Address correspondence to Dr Junxian Lim or Professor David Fairlie, Institute for Molecular

Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. Email:

j.lim@imb.uq.edu.au, d.fairlie@imb.uq.edu.au

2

Abstract Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug

discovery. It has been traditionally determined by binding putative new ligands in competition

with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a

lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability and

safety. Here, we have chemically synthesized the 77-residue human C3a protein and conjugated

its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent

protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-

DTPA-hC3a binds selectively to its cognate G protein coupled receptor C3aR with full agonist

activity and similar potency and selectivity as native C3a in inducing calcium secretion and

phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed

C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a

dissociation constant Kd 8.7 ± 1.4 nM for Eu-DTPA-hC3a, and binding affinities for hC3a (pKi

8.6 ± 0.2, Ki 2.5 nM) and C3aR ligands TR16 (pKi 6.7 ± 0.1, Ki 138 nM), BR103 (pKi 6.7 ±

0.1, Ki 185 nM), BR111 (pKi 6.3 ± 0.2, Ki 544 nM) and SB290157 (pKi 6.3 ± 0.1, Ki 517 nM)

via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-

hC3a is a novel non-radioactive probe suitable for studying ligand-C3aR interactions with

potential value in accelerating drug development for human C3aR in physiology and disease.

3

Introduction

The human complement anaphylatoxin C3a is a 77-residue protein derived from

proteolytic cleavages of the human complement protein C3 by C3 convertases. C3a mediates a

variety of proinflammatory and immunoregulatory functions through binding to its cognate G

protein-coupled C3a receptor (C3aR). C3aR is ubiquitously expressed on immune cells,

adipocytes, epithelial cells, kidneys, liver, spleen, heart and brain. Due to its extensive

distribution, C3aR is linked to physiological responses such as metabolism, inflammation and

immunity.1,2 Apart from modulating immunity, C3aR is implicated in the pathogenesis and

progression of various inflammatory diseases including asthma, diet-induced obesity, sepsis,

colitis and arthritis.3-6 However, the biological role and interaction of C3a and C3aR-mediated

disease pathologies remain to be fully elucidated. The design and development of small potent

synthetic molecules that can selectively target C3aR is required to interrogate and potentially

treat C3aR-mediated diseases.

Biophysical binding assays that interrogate C3aR-ligand interactions are essential

analytical tools to discover potent C3aR modulators. We have previously employed traditional

radioisotope binding assays using [125I]-C3a as a label to study C3aR binding affinity of a

series of small ligands capable of mimicking C3a and acting as effective antagonists or agonists

of C3aR.7,8 However, the short half-life of [125I]-C3a and restrictions associated with

manipulation of the radionuclide have limited the practicability of this assay, prompting us to

pursue alternative methods. Lanthanides such as, europium, samarium and terbium are rare-

earth metals regarded as attractive alternatives to organic fluorescent molecules and radioactive

labeling probes. They display unique fluorescence properties, such as long lifetime of

luminescence (µs-ms), which allows highly sensitive detection of biological probes in complex

environments by time-resolved fluorescence spectroscopy; a large Stokes shift (~150 nm)

which minimizes emission/excitation overlap; and narrow emission peaks (10–20 nm) which

4

collectively contribute to increase signal-to-noise ratio and sensitivity.9,10 Together with the

widely used dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) system, the

bound lanthanide on the receptor is dissociated under low pH to give a highly stable fluorescent

signal.11

Based on these advantageous analytical properties of lanthanide-based fluorophores, we

sought to develop a novel human C3a probe labeled with a Eu3+ chelate for use in time-

resolved fluorescence binding assays for the screening of C3aR interacting molecules. Herein,

we apply the native chemical ligation (NCL) approach12 to synthesize full-length human C3a

specifically modified at the N-terminus by appending diethylenetriaminepentaacetic acid

(DTPA),13 a known chelating ligand for europium (Eu-DTPA-hC3a, Figure 1). Next, we

validated its functional profile by measuring intracellular calcium release and phosphorylation

of extracellular signal-regulated kinase (ERK1/2) in pharmacological assays. We also

demonstrated saturation and competitive binding for the novel Eu-DTPA-hC3a in competition

with human C3a and known C3aR-specific small molecule ligands (TR16, BR103, BR111 and

SB290157). The results establish that Eu-DTPA-hC3a retains the same pharmacological

properties as native human C3a, inducing full C3aR activation, competitively binding to C3aR,

and competing like C3a with binding by four small molecule agonists and antagonists.

Results and Discussion

Chemical synthesis of the novel Eu-DTPA-hC3a

Human C3a is composed of 77 amino acid residues and contains three intramolecular

disulfide bonds (Figure 1). According to Ghassemian et al, synthetic access to full length linear

C3a molecule is better achieved using a fragment ligation approach due to its extended

sequence.12 Synthetic peptide segments can be readily prepared using an automated peptide

synthesizer employing standard Fmoc protected amino acids and established solid support

5

detachment procedures. We therefore sought to construct synthetic human C3a using a NCL

approach suitable for automated Fmoc-chemistry, based on sequential ligation of C-terminally

modified peptide hydrazides developed by the Liu group14. DTPA was chosen as a chelating

ligand due to its high affinity for europium, excellent solubility, and compatibility with NCL

methods and C3a folding steps.13 The positioning of the Eu-DTPA complex at the N-terminus

of human C3a was designed so as not to interfere with the key C-terminal effector or activating

region of C3a.

The human C3a sequence was divided into three peptide fragments: DTPA-C3a[1-22]-

NHNH2 (1), H-C3a[23-48]-NHNH2 (2) and H-C3a[49-77]-OH (3), which were individually

prepared on resin by standard Fmoc-SPPS using a peptide synthesizer. Fragment 1 and 2 were

assembled on a hydrazine-modified trityl resin,15 while fragment 3 was built on a 2-Cl-trityl

solid support. DTPA was attached to the N-terminus of fragment 1 using DTPA anhydride via a

previously described method.16

While fragments 1 and 3 were obtained in good yield and purity, fragment 2 could not be

readily isolated after a troublesome synthesis by Fmoc-SPPS. Therefore, we instead adopted a

Boc-SPPS method previously reported by us12 to build the second fragment with higher

efficiency, as the thioester Thz23-C3a[24-48]-COSR (4). Before NCL, the C-terminal hydrazide

of 1 was transformed into a reactive thioester upon activation with NaNO2 and reaction with

sodium 2-mercaptoethanesulfonate (MESNA)14 affording DTPA-C3a[1-22]-

COSCH2CH2SO3H thioester 5 quantitatively.

Full assembly of reduced C3a was accomplished by consecutively joining the three

polypeptides in the C- to N- direction (Figure 1). First, thioester 4 was combined with 3 under

typical NCL-based thiolysis conditions to form intermediate 6. After conversion of Thz23 to

Cys23, C3a[23-77]-OH 6 was likewise ligated to 5 affording reduced DTPA-C3a 7. Mass

6

spectroscopy analysis confirmed the formation of the 77-residue DTPA-capped peptide 7

(expected mass: 9469.0, observed mass: 9471.1, Figure S1).

N

NO

O OO

NO

O

O

OO

Eu3+

Eu-DTPA-C3a

6

Eu-DTPA-C3a = 9615.5 Da

+12

+11+10

+9

+8+7

NH

N NO

OOH

C3a [1-22] NHNH2

O

H2NHN

HO ON

HO O

OOH

1

c

HNDTPA C3a [1-22] SR'

O

5

C3a [24-48] SR

O

4

HN

S

HN C3a [50-77]H2N

O

HS

3

49

d

HN C3a [50-77]N

H O

SH

C3a [24-48]

O

HN

S

4923

23

HN C3a [24-77]N

H O

SHOHNDTPA C3a [1-22]

a, b

Cl

+

+

e, d

23f, g

7

a

Figure 1. Total chemical synthesis of Eu-DTPA-hC3a by native chemical ligation. Reaction conditions: a) i. Fmoc-AA-OH, HCTU, DIPEA in DMF; ii. 33% piperidine in DMF; several cycles. b) DTPA anhydride, DMSO, overnight. c) i. NaNO2, 6 M guanidine, 0.2 M phosphate buffer pH 3.0, -15 °C, 20 min; ii. MESNA, 6 M guanidine, 0.2 M phosphate buffer pH 7.0, room temperature, 5 min. d) 50 mM TCEP, 50 mM MPAA, 6 M guanidine, 0.2 M phosphate buffer pH 7.0, 5-6 h. e) methoxyamine HCl, pH 3.5; f) 8 mM reduced glutathione, 1 mM oxidized glutathione, 0.6 M guanidine, 50 mM phosphate buffer pH 7.5, room temperature, 2 h. g) EuCl3, 0.1 M ammonium acetate buffer pH 7.5, overnight. R = CH2CH2CO-Arg, R’ = CH2CH2SO3H. Bottom: mass spectrum of pure synthetic Eu-DTPA-hC3a; expected mass: 9614.9, observed mass 9615.5.

Folding of the C3a derivative was carried out in the presence of a glutathione redox

system12 to give oxidized DTPA-C3a 8 (MS: expected mass: 9463.0, observed mass: 9464.8,

Figure S2). Finally, Eu3+ complexation was achieved by treating peptide 8 with three

7

equivalents of EuCl3 in ammonium acetate buffer to give pure folded Eu-DTPA-hC3a (Figure

1). Europium complexation was appropriately performed as the last step of the synthesis, since

europium chelation is not compatible with acidic RP-HPLC conditions.

Pharmacological validation of Eu-DTPA-hC3a as a C3aR agonist

To characterize the potency and specificity of synthetic Eu-DTPA-hC3a, concentration-

dependent intracellular calcium mobilization was determined on HEK293 cells stably

transfected with human Gα16 and human C3aR (HEK293 Gα16-C3aR). Gα16 belongs to the Gαq

protein family, which mediates the activation of phospholipase C leading to the subsequent

inositol triphosphate-stimulated mobilization of intracellular calcium from the endoplasmic

reticulum.17 Gα16 proteins are widely used in high-throughput fluorometric calcium imaging

assays for the screening of new GPCR ligands.18 Furthermore, we have previously

characterized C3aR ligands using intracellular calcium mobilization7,8 and previous studies

have also overexpressed Gα16 to examine the functional activity of human C3aR using an

intracellular calcium mobilization assay.19

Based on concentration-dependent curves, Eu-DTPA-hC3a (pEC50 8.2 ± 0.1, EC50 6 nM)

and C3a (pEC50 8.5 ± 0.1, EC50 3 nM) demonstrated very similar full agonist activity and

comparable potency in inducing C3aR-mediated intracellular calcium mobilization (Figure

2A). Hence, the addition of Eu-DPTA to the N-terminus of C3a did not interfere with the

ability of the protein to act as a full agonist at C3aR. To establish whether Eu-DTPA-hC3a was

specific for C3aR, and that the intracellular calcium mobilization was not an off-target

activation of other endogenous GPCRs, both Eu-DTPA-hC3a and C3a were tested on HEK293

cells transfected with empty vector (HEK293 Gα16 cells). Even at a high concentration of 300

nM, Eu-DTPA-hC3a failed to induce any intracellular calcium mobilization in HEK293 Gα16

cells (Figure 2B). These results indicate that the novel conjugate Eu-DTPA-hC3a is a full

8

agonist in mediating intracellular calcium mobilization, while retaining its native specificity for

human C3aR.

Figure 2. Eu-DTPA-hC3a mediates intracellular calcium mobilization via C3aR in HEK293 cells transfected with both human Gα16 and human C3aR (HEK293 Gα16-C3aR). (A) Concentration-dependent curves of intracellular calcium mobilization by Eu-DTPA-hC3a versus C3a on HEK293 Gα16-C3aR cells. Eu-DTPA-hC3a (pEC50 8.2 ± 0.1, EC50 6 nM) showed similar full agonist activity compared to C3a (pEC50 8.5 ± 0.1, EC50 3 nM). (B) Eu-DTPA-hC3a and C3a failed to induce intracellular calcium mobilization in HEK293 cells transfected with empty vector (HEK293 Gα16). Calcium responses were expressed as a percentage of the maximum response induced by 300 nM human C3a on HEK293 Gα16-C3aR cells. Error bars represent mean ± SEM of > 3 independent experiments.

To further validate the retention of C3a-like signaling properties of Eu-DTPA-hC3a in

mediating C3aR activation, phosphorylation of ERK1/2 in HEK293 Gα16-C3aR cells was also

analyzed. A temporal profile was first established by treating HEK293 Gα16-C3aR cells with

Eu-DTPA-hC3a (100 nM) or C3a (100 nM) at different time points, as ERK1/2

phosphorylation can be rapid or delayed depending on the nature of the ligand, cell type,

9

receptor and signaling pathway being examined.20 Both Eu-DTPA-hC3a and hC3a showed

similar temporal profiles, with maximum phosphorylation of ERK1/2 at 3 min before returning

to basal levels at 120 min (Figure 3A).

Figure 3. Eu-DTPA-hC3a displays similar temporal and potency profiles as native human C3a in mediating phosphorylation of ERK1/2 (p-ERK1/2) in HEK293 Gα16-C3aR cells. (A) HEK293 Gα16-C3aR cells were treated with 100 nM Eu-DPTA-hC3a or C3a at indicated time points. Phosphorylation of ERK1/2 was expressed as a percentage of the maximum response at 3 min. (B) Stimulation with Eu-DTPA-hC3a or C3a for 3 min mediates phosphorylation of ERK1/2 in a concentration-dependent manner. Eu-DTPA-hC3a (pEC50 8.8 ± 0.1, EC50 1 nM) showed similar full agonist activity as compared to hC3a (pEC50 9.0 ± 0.1, EC50 0.9 nM). Phosphorylation of ERK1/2 was expressed as a percentage of the maximum response induced by 300 nM hC3a. Error bars represent mean ± SEM of n > 3 independent experiments.

Concentration-dependent curves of Eu-DTPA-hC3a versus C3a also demonstrated similar

agonist profiles in C3aR-mediated ERK1/2 phosphorylation (Figure 3B). Eu-DTPA-hC3a

(pEC50 8.8 ± 0.1, EC50 1 nM) showed similar full agonist activity as C3a (pEC50 9.0 ± 0.1, EC50

0.9 nM). Consistent with the results from the intracellular calcium mobilization assay (Figure

2), Eu-DTPA-hC3a exhibits similar agonist and temporal profiles as C3a in inducing

phosphorylation of ERK1/2. These results further confirmed that incorporating the Eu-DPTA

10

complex onto the N-terminus of human C3a does not interfere with potency, selectivity or

pharmacological properties mediated by C3a through its receptor human C3aR.

Saturation and competitive binding of C3aR using the novel Eu-DTPA-hC3a

Ligand affinity plays a critical role in GPCR drug design and development,21 but in the

case of C3aR, ligand-receptor affinity measurements have required use of the radioligand [125I]-

C3a.7,8 Lanthanide-based fluorescence studies have been developed for other GPCRs, such as

CXC chemokine receptor 1, CXC chemokine receptor 2, neurokinin 1 receptor, neurokinin 2

receptor, neurotensin receptor, β2-adrenergic receptor, δ-opioid receptor, relaxin/insulin like

family peptide receptor 2, melanocortin-4 receptor and protease-activated receptor 2.22-26 To

test the applicability of Eu-DTPA-hC3a as a labeled ligand for C3aR affinity, saturation

binding experiments were performed on HEK293 Gα16-C3aR cells (Figure 4). Eu-DTPA-hC3a

was found to bind in a saturable and specific manner to C3aR, with a calculated Kd 8.7 ± 1.4

nM when fitted to a one-site binding model. The non-specific binding was determined to be <

15% of the total binding at the highest concentration of Eu-DTPA-hC3a (100 nM).

0 25 50 75 1000

50

100

150

Eu-DTPA-C3a (nM)

% b

indi

ng o

fE

u-D

TPA

-C3a

Total Non-specific

Figure 4. Saturation binding curves of Eu-DTPA-hC3a to HEK293 Gα16-C3aR cells. For total binding, cells were incubated with increasing concentrations of Eu-DPTA-hC3a for 60 min at room temperature with shaking. Non-specific binding was determined in the presence of 5 µM unlabeled C3a. The calculated dissociation constant (Kd) for Eu-DTPA-hC3a was 8.7 ± 1.4 nM. Error bars represent mean ± SEM of n > 3 independent experiments.

11

To demonstrate Eu-DTPA-hC3a as a fluorescence non-radioactive probe for C3aR in a

physiological context, primary human peripheral blood mononuclear cells (PBMC) from buffy

coats were used for competitive binding assays with human C3a (IC50 1.2 nM– 2.7 nM) (Figure

5A and Figure S3). The specificity of Eu-DTPA-hC3a to C3aR was again confirmed using

HEK293 Gα16-C3aR and HEK293 Gα16 cells in a competitive binding assay with C3a (Figure

5B). HEK293 Gα16-C3aR cells yielded a typical sigmoidal competitive curve with Ki 2.5 nM,

while < 10% of Eu-DTPA-hC3a was bound on HEK293 Gα16 cells (Figure 5B). Consistent

with the intracellular calcium mobilization assay (Figure 2), Eu-DTPA-hC3a is specific and

requires C3aR for binding to the cells and for intracellular signaling.

12

-12 -10 -8 -6

0

5000

10000

15000

Log [C3a] (M)

Eu-D

TPA

-C3a

(337

/620

nm) HEK293 Gα16-C3aR

HEK293 Gα16

-10 -8 -6 -4

0

5000

10000

15000

Log [Ligand] (M)

Eu-D

TPA

-C3a

(337

/620

nm)

TR16 BR013SB290157 3D53

BR111

B

C

A

-12 -10 -8 -6

0

1000

2000

3000

4000

Log [C3a] (M)

Eu-D

TPA

-C3a

(337

/620

nm)

PBMC

Figure 5. Competitive binding assay of Eu-DTPA-hC3a with C3a, selective C3aR ligands (TR16, BR103, BR111, SB290157) or selective C5aR ligand (3D53). Competitive binding experiments were performed on (A) primary human peripheral blood mononuclear cells (PBMC), (B) HEK293 Gα16-C3aR and HEK293 Gα16 cells using a single concentration of Eu-DTPA-hC3a (2 nM) in the presence of increasing concentrations of human C3a. Data for primary human PBMC are shown from one representative donor from four donors. (C) Competitive binding experiments were performed on HEK293 Gα16-C3aR cells using a single concentration of Eu-DTPA-hC3a (2 nM) in the presence of increasing concentrations of C3aR or C5aR ligand. Cells were incubated with Eu-DTPA-hC3a and C3aR or C5aR ligand for 60 min at room temperature with shaking. Binding affinities of C3a (pKi 8.6 ± 0.2, Ki 2.5 nM), TR16 (pEC50 6.7 ± 0.1, Ki 138 nM), BR103 (pKi 6.7 ± 0.1, EC50 185 nM), BR111 (pKi 6.3 ± 0.2, Ki 544 nM), SB290157 (pKi 6.3 ± 0.1, Ki 517 nM) and 3D53 (pKi >4.5, Ki >30 µM) as measured by the displacement of Eu-DTPA-hC3a (2 nM). Error bars represent mean ± SEM of ≥ 3 independent experiments.

13

Eu-DTPA-hC3a was also additionally validated in competitive binding experiments with

known C3aR-specific ligands (TR16,7 BR103,27 SB29015728 and BR11129) (Figure S4) on

HEK293 Gα16-C3aR cells (Figure 5C). The Ki values for TR16, BR103, BR111 and SB290157

were 138 nM, 185 nM, 544 nM and 514 nM, respectively. As a control, the C5aR specific and

potent antagonist, 3D53, which does not bind to or activate C3aR30-33 showed no competitive

binding with Eu-DTPA-hC3a even at the highest concentration of 30 µM. We have previously

reported competitive binding IC50 values for C3a (0.07 nM), TR16 (4 nM) and SB290157 (10

nM) against 80 pM of [125I]-C3a in primary human monocyte-derived macrophages.7,34 The

differences in the competitive binding affinities observed for C3a and TR16 against the

differently labeled Eu-DTPA-hC3a and [125I]-C3a in these two different cell lines could be

explained by different expression levels of C3aR on transfected HEK293 Gα16-C3aR cells or

the higher concentration of Eu-DTPA-hC3a compared to [125I]-C3a or the different cell types

used. Despite those differences, it was noted that Eu-DTPA-hC3a displayed consistent and

expected rank order competitive binding affinities (C3a > TR16 > BR103 > BR111 >

SB290157) that were in the same order as found by [125I]-C3a affinity binding.

It is estimated that about 30% of marketed drugs target GPCRs or GPCR-mediated

mechanisms.35 The evaluation of the ligand-receptor affinity using labeled probes is therefore

essential for drug development and discovery. An ideal label should not alter the intrinsic

bioactivity and pharmacological properties of the ligand, should be cost-effective, easy to

operate and offer good signal-to-noise ratio. Radioisotopes such as 125I or 3H are widely used as

labels without significantly affecting binding properties of the ligand, however the high cost,

limited shelf life due to isotope decay, limitations on how they are used, and potential health

hazards, are drawbacks of radiolabeled ligand binding assays. The use of lanthanide-based

14

fluorophores, on the other hand, overcomes most of these disadvantages, offering a cost-

effective and user-friendly probe with a considerably enhanced signal-to-noise ratio.36

In conclusion, this study has successfully developed a novel Eu-DTPA-hC3a

bioconjugate using chemical synthesis. Human C3a protein was synthesized and specifically

conjugated at the N-terminus with a Eu-DTPA complex. The DTPA chelator was unaltered

during all steps of synthesis and did not interfere with oxidative protein folding, allowing

formation of a functionally active C3a protein that behaved as a full agonist at the C3a receptor.

The novel Eu-DTPA-hC3a bioconjugate showed no discernable differences in potency,

specificity and pharmacological profiles compared to native C3a. The development of this non-

radioisotope alternative for a C3aR-ligand binding assay can help confirm hC3aR binding of

other short peptides, such as casoxin C, oryzatensin and TLQP-21 which have been reported to

be ligands for C3aR.37-40 It could also potentially accelerate the design and development of

potent and selective small molecules to elucidate and modulate C3aR-mediated physiology and

disease pathology.

Experimental Procedures

Solid-phase peptide synthesis (SPPS)

The human C3a sequence was assembled from three synthetic segments: DTPA-C3a[1-

22]-NHNH2 (1), Thz23-C3a[24-48]-COS-CH2CH2CO-Arg (4)12 and H-C3a[49-77]-OH (3).

Peptides 1 and 3 were synthesized on solid support by Fmoc-based SPPS on an automated

peptide synthesizer (Symphony, Protein Technologies) using a standard HCTU/DIPEA

activation protocol and Fmoc-protected amino acids.41 Hydrazide-modified peptide 1 was

assembled on a freshly prepared hydrazine-trityl resin, which was obtained from hydrazination

of 2-Cl-trityl resin as previously described.15 Fragment 3 was assembled directly on a 2-Cl-

15

trityl resin. Coupling of DTPA to the N-terminus of the C3a[1-22] segment was accomplished

by treating the dry free-amine peptide-bound resin overnight with a mixture of DTPA

anhydride (4 eq) and 1-hydroxybenzotriazole (8 eq) in anhydrous DMSO (preheated to allow

reagent dissolution and allowed to react for 30 min before addition to resin).16 Peptide

segments 1 and 3 were cleaved from the solid support by treatment with a cocktail of

TFA:triisopropylsilane:water:phenol (88:2:5:5) for 3 h, precipitated in ice-cold diethyl ether,

redissolved in a aqueous solution containing 50% acetonitrile/0.01% TFA and lyophilized.

Crude peptides were purified by reversed phase high performance liquid chromatography (RP-

HPLC) using a C18 column (Phenomenex Luna 10 µm, 100 Å, 250 x 21 mm,) eluting at a flow

rate of 20 mL/min and a gradient of 0 to 40% buffer B (90% CH3CN/10% H2O/0.1% TFA) in

buffer A (0.1% TFA in water) over 40 min. Analytical RP-HPLC and ESI-MS were used to

confirm the purity and molecular mass of the synthetic peptides (Table S1, Figure S5).

Thioester 5. Hydrazide 1 was dissolved at 5 mM concentration in 0.2 M phosphate

buffer pH 3.0 containing 6 M guanidine.HCl and cooled to -15 0C using an ice/salt bath.

NaNO2 (50 eq) dissolved in minimum water was added dropwise and the resulting mixture was

stirred at -15 0C for 20 min. MESNA (200 eq) was dissolved at 70 mg/mL concentration in 0.2

M phosphate buffer pH 7.0/6M guanidine.HCl and the pH was readjusted to 7 by adding

aqueous NaOH. The MESNA solution was then added dropwise to the cold peptide reaction

and the resulting mixture was stirred at room temperature for a further 5 minutes. The reaction

was quenched by addition of 1% TFA and the resulting thioester 5 was purified by RP-HPLC

using a C18 Luna Phenomenex column. MS for 5: found 3136.8, expected 3136.7 Da (Figure

S5).

Assembly of linear DTPA-hC3a (7) by native chemical ligation

16

Ligation of 4 and 3. Thioester 4 (1 eq) and peptide fragment 3 (1 eq) were dissolved in

NCL buffer (50 mM TCEP, 50 mM MPAA, 6 M guanidine.HCl, 0.2 M phosphate buffer pH

7.0) to a 2 mM peptide concentration and the reaction was stirred at room temperature for 5 h.

After that, solid TCEP was added (approximately same mg amount of peptide), the reaction

was stirred for further 20 min and finally quenched by addition of 1% TFA. Intermediate 6 was

purified by RP-HPLC using a Agilent Zorbax 300SB C18 column as previously described.12

MS for 6: found 6488.2, expected 6487.6 Da (MS and HPLC data in accordance with ref. 12).

Ligation of 6 and 5. Conversion of Thz23 to Cys23 in peptide 6 was accomplished by

treating 6 with methoxyamine as previously described.12 The resulting intermediate H-C3a[23-

77]-OH was then ligated to 5 using the same conditions as described for the ligation of 4 and 3.

The resulting full-length reduced DTPA-C3a 7 was purified by RP-HPLC using a Agilent

Zorbax 300SB C18 column as previously described.12 MS for 7: found 9471.1, expected 9470.0

Da (Figure S1).

Oxidative folding of DTPA-hC3a

Oxidation of DTPA-hC3a peptide 7 was performed as described.12 Briefly, 7 was

dissolved in a minimum volume of 6 M guanidine.HCl and combined with an argon-degassed

oxidation buffer containing 50 mM Na2HPO4, 8 mM reduced L-glutathione, 1 mM oxidized L-

glutathione, pH 7.5. After 2 h reaction, oxidized DTPA-C3a was purified by RP-HPLC using

an Agilent Zorbax 300SB C18 column. MS for oxidized DTPA-hC3a 8: found 9463.7 Da;

expected 9464.0 Da (Figure S2).

Europium chelation

Oxidized DTPA-hC3a 8 was dissolved in 0.1 M ammonium acetate buffer pH 8.0 at 200

µM concentration and combined with the same volume of 600 µM EuCl3 (in 0.1 M ammonium

17

acetate buffer) and the resulting solution was stirred at room temperature overnight. Unbound

Eu3+ was removed by solid phase extraction using an Oasis HLB cartridge using the

manufacturer instructions. The eluted pure Eu3+-labeled peptide was then lyophilized to yield

an amorphous white powder. Full conversion to lanthanide-labeled Eu-DTPA-hC3a was

confirmed by measuring the mass spectrum of an analytical sample dissolved in acid free 50%

acetonitrile and directly injected into the mass spectrometer. A single mass spectrum

corresponding to Eu-DTPA-hC3a was found (deconvoluted mass: 9615.5, expected 9614.9;

Figure 1).

Other C3aR and C5aR ligands

C3aR ligands (TR167, BR10327, BR11129 and SB29015728) and C5aR ligand (3D5331)

were synthesized and characterized as previously described (Figure S4).

Cell culture

Cell culture reagents were purchased from Invitrogen. Human embryonic kidney

(HEK)293 cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with

10% fetal bovine serum and 50 units/mL of penicillin-streptomycin at 37°C in a 5% CO2

incubator. Human full-length wild-type Gα16 and C3aR plasmids were purchased from cDNA

Resource Center and Sino Biological respectively. HEK293 cells were transfected using

Lipofectamine® 3000 Reagent to coexpressed Gα16 and C3aR. Stable clones were selected in

the presence of hygromycin B (300 µg/mL) and geneticin (750 µg/mL). Primary human PBMC

were isolated from buffy coat (Australian Red Cross Blood Service) and used for binding

experiments on the same day. PBMC were harvested and purified using Ficoll-Paque PLUS

(GE Healthcare) density centrifugation.

18

Intracellular calcium mobilization assays

Intracellular calcium mobilization assays were performed as previously described.42

HEK293 Gα16-C3aR or HEK293 Gα16 cells were seeded at a density of 5,000 cells per well and

allowed to adhere overnight. Various concentrations of C3a or Eu-DTPA-hC3a were added via

a fluorescent imaging plate reader (FLIPR, Molecular Devices) and intracellular calcium

mobilization was monitored via fluorescence measurement for 300 s (excitation 470-495 nm,

emission 515-575 nm).

Phosphorylation of ERK1/2 assays

HEK293 Gα16-C3aR cells were seeded at a density of 10,000 cells per well and allowed

to adhere overnight. Cells were serum-starved for 3 h prior to experiment and dilution of Eu-

DTPA-hC3a and C3a were performed in serum-free medium. For temporal analysis, HEK293

Gα16-C3aR cells were treated with 100 nM Eu-DTPA-hC3a or hC3a at 1, 3, 5, 10, 15, 30, 60 or

120 min for 37°C. For concentration-dependent analysis, HEK293 Gα16-C3aR cells were

treated with various concentration of Eu-DTPA-hC3a or hC3a for 3 min at 37°C.

Phosphorylation of ERK1/2 was quantified using AlphaLISA® SureFire® Ultra p-ERK1/2

Assay Kit (PerkinElmer) according to manufacturer’s instructions.

Saturation and competitive binding assays

HEK293 Gα16-C3aR or HEK293 Gα16 cells were non-enzymatically lifted using Versene

Solution. Cells were then resuspended in 2% bovine serum albumin (BSA) in phosphate

buffered saline (PBS) and seeded at 30,000 cells per well in a round-bottom 96 well plate. All

dilutions of Eu-DTPA-hC3a, hC3a and ligands were performed in PBS supplemented with 2%

BSA. Saturation binding were tested using increasing concentration of Eu-DTPA-hC3a and

non-specific binding was performed in the presence of 5 µM C3a. For competitive binding

19

experiments, primary human PBMC (600,000 cells per well) or HEK293 Gα16-C3aR cells

(30,000 cells per well) were simultaneously treated with Eu-DTPA-hC3a (2 nM) and various

concentrations of hC3a or C3a ligand for 60 min at room temperature with shaking. Cells were

then washed thrice with PBS supplemented with 0.2% BSA, 20 µM EDTA and 0.01% Tween-

20 by repeated centrifugation. After washings, cells were re-suspended with 20 µL of DELFIA

enhancement solution (PerkinElmer) and then transferred to a white 384-well ProxiPlate

(PerkinElmer) to incubate for 90 min at room temperature. Time-resolved fluorescence was

measured using PHERAstar plate reader (BMG Labtech) at 337 nm excitation followed by 400

µs delay before 620 nm emission.

Data analysis

Data were plotted and analyzed using GraphPad Prism 7 for Mac OS X. EC50 and Ki

values were calculated using nonlinear regression four parameters dose-response curve and one

site competitive binding model respectively. All values of independent parameters are shown as

mean ± SEM of least three independent experiments (n ≥ 3) unless otherwise stated.

Acknowledgements

This research was supported by the National Health and Medical Research Council

(grant 1084018; Senior Principal Research Fellowships 1027369, 1117017), the Australian

Research Council (grant DP1030100629), the Australian Research Council Centre of

Excellence in Advanced Molecular Imaging (CE140100011) and the Australian Red Cross

for human PBMC isolation from buffy coats.

Supporting Information

20

Characterization data (analytical HPLC and MS) of peptide fragments, intermediates

and full length Eu-DTPA-hC3a. Competitive binding data on primary human PBMC.

Chemical structures of TR16, BR111, BR103, SB290157 and 3D53.

Abbreviations

BSA bovine serum albumin; C3aR C3a receptor; DELFIA dissociation-enhancer

lanthanide fluorescence immunoassay; DIPEA diisopropylethylamine; DTPA

diethylenetriaminepentaacetic acid; ERK extracellular signal-regulated kinases; Eu europium;

FLIPR fluorescent imaging plate reader; GPCR G protein-coupled receptor; HCTU (2-(1H-6-

chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate); HEK human

embryonic kidney; MPAA 50 mM 4-mercaptophenylacetic acid; MESNA Sodium 2-

mercaptoethanesulfonate; PBMC peripheral blood mononuclear cells; PBS phosphate buffered

saline. RP-HPLC, reversed phase high performance liquid chromatography; TFA

trifluoroacetic acid; TCEP tris(2-carboxyethyl)phosphine.

Conflict of Interest Disclosure

The authors declare no competing financial interest in this publication.

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27

TOC graphic

N

NO

O OO

NO

O

O

OO

Eu3+

-10 -8 -6 -4

0

5000

10000

15000

Log [Ligand] (M)

Eu-

DTP

A-C

3a(3

37/6

20nm

) TR16 BR013SB290157 3D53

BR111

Eu-DTPA-C3a