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. 2021 Nov 17;4(6):1808–1817. doi: 10.1021/acsptsci.1c00199

Development of Synthetic Human and Mouse C5a: Application to Binding and Functional Assays In Vitro and In Vivo

Declan M Gorman †, Xaria X Li †, Colton D Payne †, Cedric S Cui †, John D Lee †, K Johan Rosengren †, Trent M Woodruff †,‡, Richard J Clark †,§,*
PMCID: PMC8669711  PMID: 34927012

Abstract

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The complement activation peptide C5a is a key mediator of inflammation that is associated with numerous immune disorders. C5a binds and activates two seven-transmembrane receptors, C5aR1 and C5aR2. Experimentally, C5a is utilized to investigate C5a receptor biology and to screen for potential C5aR1/C5aR2 therapeutics. Currently, laboratory sources of C5a stem from either isolation of endogenous C5a from human serum or most predominantly via recombinant expression. An alternative approach to C5a production is chemical synthesis, which has several advantages, including the ability to introduce non-natural amino acids and site-specific modifications whilst also maintaining a lower probability of C5a being contaminated with microbial molecules or other endogenous proteins. Here, we describe the efficient synthesis of both human (hC5a) and mouse C5a (mC5a) without the need for ligation chemistry. We validate the synthetic peptides by comparing pERK1/2 signaling in CHO-hC5aR1 cells and primary human macrophages (for hC5a) and in RAW264.7 cells (for mC5a). C5aR2 activation was confirmed by measuring β-arrestin recruitment in C5aR2-transfected HEK293 cells. We also demonstrate the functionalization of synthetic C5a through the introduction of a lanthanide chelating cage to facilitate a screen for the binding of ligands to C5aR1. Finally, we verify that the synthetic ligands are functionally similar to recombinant or native C5a by assessing hC5a-induced neutrophil chemotaxis in vitro and mC5a-mediated neutrophil mobilization in vivo. We propose that the synthetic hC5a and mC5a described herein are valuable alternatives to recombinant or purified C5a for in vitro and in vivo applications and add to the growing complement reagent toolbox.

Keywords: C5a, solid-phase peptide synthesis, complement, receptor binding

Native human C5a (hC5a) is a 74-amino acid, glycosylated peptide, cleaved from a 195 kDa precursor protein known as C5, while mouse C5a (mC5a) is 77-amino acid, nonglycosylated peptide, cleaved from its precursor C5 protein (189 kDa) referred to as Hc.1,2 C5a has two endogenous receptors, C5aR1 and C5aR2. C5aR1 has been well characterized; however, its second receptor, C5aR2, is less well understood.3,4 C5a, via its interaction and activation of C5aR1, is a potent pro-inflammatory mediator, contributing to host defense against invading pathogens but also sustained inflammatory responses. These sustained responses are thought to lead to an exacerbation of a myriad of immune and inflammatory sterile and infectious conditions.5,6 As a result, antagonist compounds targeting the C5a-C5aR1 interaction for treatments of these indications are being actively pursued.7−9 Similarly, the mechanisms underlying C5a-induced C5aR2 activation are currently being investigated for ischemic, traumatic, and infections tissue injuries.10−12

Currently, the most common methods of obtaining human and mouse C5a for research purposes include recombinant expression in bacteria (rhC5a and rmC5a) and isolation of native hC5a from human blood. Chemical synthesis offers an alternative approach to the procurement of C5a with several advantages, including the incorporation of non-natural amino acids and site-selective chemical modifications. The chemical synthesis of hC5a using a two fragment ligation method has recently been reported; however, this approach introduces an extra level of complexity compared to standard on-resin synthesis of the full sequence.13 hC5a has seven cysteine residues, six of which form three disulfide bonds, while the extra cystine (Cys27) is partially cysteinylated (disulfide bonded to a free cysteine residue).14 This complicates the folding of hC5a, as unreacted cysteines can result in the formation of disulfide-linked dimers. It has previously been described that Cys27 can be mutated without adversely affecting receptor binding.15 In fact, phage display experiments involving C5a-desArg have revealed that the mutation of Cys27 to Arg is consistently incorporated in analogues with improved binding to C5aR1.16,17 Despite these reports, evidence on how this change affects the signaling activity of hC5a is lacking in the literature.

Full-length human and mouse C5a is rapidly cleaved in vivo by carboxypeptidases into C5a-desArg, a stable form of C5a with a reduced potency at C5aR1.18,19 Interestingly, hC5a-desArg was observed to bind with a higher affinity to C5aR2 than C5aR1.20 It has also been reported that hC5a-desArg has an altered scope of bioactivity to that of hC5a. This is exemplified by its inability to induce late continuous leukotriene C4 production from basophils, a process that is induced by hC5a.21 Overall, these properties make C5a-desArg an interesting molecule to study further. However, while the chemical synthesis of rat-C5a-desArg via a ligation approach has been described, a strategy for the on-resin synthesis of hC5a-desArg has not yet been outlined.22 Similarly, the synthesis of mC5a has also not yet been described in the literature. Considering the widespread use of mouse models of complement mediated inflammation and disease, its utility as an in vivo tool of mouse-specific C5aR1/C5aR2 activity is notable.12,23−26

The development of potential therapeutics targeting C5aR1 and C5aR2 rely on being able to screen both receptor activation along with receptor binding with high-throughput capacity. There are currently multiple approaches of high-throughput screening of C5aR1 activation, including pERK1/2 signaling and calcium mobilization assays.27 However, the probing of C5aR2 activation currently relies on experiments such as β-arrestin BRET assays, which have a limited throughput capacity. In addition, activation and binding are rarely analogous, which requires activation assay results to be cross-referenced with binding assays for both C5aR1 and C5aR2.

The classical method of probing C5aR1/C5aR2 binding utilizes [I125]-C5a; however, there are many drawbacks associated with this approach, including complications from working with radioisotopes to a relatively short shelf life of labeled ligands. Lanthanide-based binding assays provide an alternative approach through the incorporation of lanthanide chelates such as diethylenetriaminepentaacetic acid (DTPA, or pentetic acid) into the sequence of the desired ligand. Similar to [I125], lanthanide chelates can be introduced without significantly affecting the binding properties of ligands to their respective receptors. However, in contrast to the use of radioisotopes, lanthanides are safe to work with, have a long shelf life, and can be utilized in high-throughput screens. The creation of a lanthanide labeled C3a analogue has previously been described;28 however, the development of a lanthanide labeled C5a analogue is notably absent both commercially and in the literature.

In this work, we describe the first efficient chemical synthesis, folding, and isolation of hC5a and mC5a without the need for ligation chemistry. We subsequently investigate whether the cysteinylation at Cys27 was important for the structure/function of hC5a by synthesizing [Ser27, Nle70]hC5a and assessing its pERK1/2 at C5aR1 and β-arrestin signaling response at C5aR2, before probing its structure using NMR spectroscopy. We also report the synthesis of synthetic [Ser27, Nle70]hC5a-desArg and compare its pERK1/2 signaling response to recombinant hC5a-desArg at C5aR1 in human monocyte-derived macrophages (HMDMs). Based on the successful synthesis of hC5a, we subsequently produce lanthanide functionalized hC5a analogues to facilitate the development of C5aR1/C5aR2 fluorescence binding assays. Finally, we verify the functionality of both synthetic mC5a and [Ser27, Nle70]hC5a by assessing neutrophil migration in vivo in mice or in human blood, respectively.

Results and Discussion

Synthesis, Oxidative Folding, and Characterization

We chose to adopt a synthetic strategy in which the entire peptide chain of hC5a was assembled on-resin, which removed the need for a ligation step as previously described.13 In addition, we substituted Met70 for an isosteric norleucine ([Nle70]hC5a) in all analogues to avoid the possibility of methionine oxidation.29 A previous study reported that the mutation of Met70 to similar residues (Leu, Val, and Ile) is tolerated as determined by both receptor binding and calcium mobilization.30 We also synthesized an analogue, [Ser27, Nle70]hC5a, where the cysteine not involved in an intramolecular disulfide bond was replaced with a serine. The main metabolite of hC5a, hC5a-desArg (with the C-terminal arginine removed), is also widely studied, and so we produced the synthetic analogue of this peptide ([Ser27, Nle70]hC5a-desArg) using the same approach as full-length hC5a (Figure S1). Although hC5a is natively glycosylated at Asn64, this modification was not incorporated in peptides synthesized in this work. Notably, rhC5a also lacks this native glycosylation. To complement the hC5a analogues, we also synthesized mC5a as it is commonly used for many C5a-related in vivo mouse studies.

In our initial attempts at synthesis using standard synthesis protocols (e.g., ∼1 mmol/g resin loading and double coupling of β-substituted residues), we observed some peptides up to Cys21 (54 residues coupled), but no substantial product was observable following this point. To address this, we used low-loaded 2-chloro-trityl chloride-linked resin (0.2 mmol/g) to reduce the likelihood of peptide aggregation during chain elongation. In addition, double coupling was implemented for every residue following Cys20 to the N-terminus, with twice the coupling reagent and amino acid equivalency. Fmoc-Gly(Dmb)-OH was used for coupling at Gly25, as initial synthesis attempts without this modified amino acid led to aspartimide formation with the subsequent Asp residue, which was inseparable from the desired product following purification. This approach led to the successful synthesis of all C5a analogues (Tables 1 and S1).

Table 1. Human and Murine C5a Analogues Sequences and Synthesis Yieldsa.

peptide sequenceb yield (%)
rhC5a NH2-MLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLGR-OH  
[Nle70]hC5a NH2-TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDXQLGR-OH 6.67
[Ser27, Nle70]hC5a NH2-TLQKKIEEIAAKYKHSVVKKCCYDGASVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDXQLGR-OH 10.11
[Ser27, Nle70]hC5a-desArg NH2-TLQKKIEEIAAKYKHSVVKKCCYDGASVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDXQLG-OH 6.74
Eu-DTPA-[Nle70]hC5a [Eu]Dtpa-TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDXQLGR-OH 0.20
Eu-DTPA-[Ser27, Nle70]hC5a [Eu]Dtpa-TLQKKIEEIAAKYKHSVVKKCCYDGASVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDXQLGR-OH 0.88
mC5a NH2-NLHLLRQKIEEQAAKYKHSVPKKCCYDGARVNFYETCEERVARVTIGPLCIRAFNECCTIANKIRKESPHKPVQLGR-OH 0.54
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a

Yield calculated from the final amount of pure peptide divided by maximum theoretical synthesis yield based on resin loading. Intramolecular disulfide-bonded cysteines are highlighted in bold. Position 27, which has a nonintramolecular bonded cysteine or a serine substitution, is underlined. Cys27 is partially cysteinylated.

b

X denotes norleucine.

Following the synthesis and initial RP-HPLC purification of the peptide chain, disulfide bonds were formed by incubating the peptide in oxidation buffer (0.1 M ammonium bicarbonate; pH 8) at a concentration of 0.2 mg/mL for 48 h. Peptides were dissolved in minimal 50/50 buffer A/B (∼5 mg/mL; 45% ACN and 0.05% TFA in H2O) before being added dropwise to the oxidation buffer. For the oxidation of [Nle70]hC5a, 0.3 mM cysteine and 3 mM cystine were added to the oxidation buffer to facilitate the partial cysteinylation of Cys27, to mirror that of native C5a. Briefly, 1 M HCl (1 mL per 10 mL of oxidation buffer) was used to quench reactions prior to purification. Following oxidation, all peptides were purified, and single isomers were isolated and identified using ESI-MS and MALDI-MS (Figures 1, S3, and S4 and Table S2). A time-course of [Ser27, Nle70]hC5a found that a 48-h oxidation was necessary to convert all linear starting materials to oxidized products (Figure S5).

Figure 1.

Figure 1

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Confirmation of synthetic hC5a and mC5a synthesis and purification by MALDI-MS and analytical RP-HPLC. (A) MALDI-MS spectrum and RP-HPLC trace of [Nle70]hC5a. Observed molecular weight: 8369.02 Da. Sample >99% pure. (B) MALDI-MS spectrum and RP-HPLC trace of [Ser27, Nle70]hC5a. Observed molecular weight: 8232.25 Da. Sample >99% pure. (C) MALDI-MS spectrum and RP-HPLC trace of [Ser27, Nle70]hC5a-desArg. Observed molecular weight: 8078.70 Da. Sample >99% pure. (D) MALDI-MS spectrum and RP-HPLC trace of mC5a. Observed molecular weight: 8897.19 Da. Sample >95% pure. * denotes peaks detected in the blank sample. All analytical traces were run at an increasing gradient of 1% buffer B (90% ACN, 0.045% TFA) in buffer A (0.05% TFA) per minute using an Agilent 300 Å, 5 μm, 150 × 2.1 mm C18 column.

Past studies have revealed that the removal of the oligosaccharide from full-length hC5a at Asn64 does not adversely affect either chemotactic or enzyme-releasing activity; however, its removal in hC5a-desArg increases activity between 5- and 10-fold compared to glycosylated hC5a-desArg.31 Although the effect of the absence of the N-linked glycosylation of Asn64 in hC5a and hC5a-desArg was not examined in this study, the reported synthesis strategy of hC5a outlined in this paper may allow for future investigation of this difference. Strategies for the incorporation of Asn-linked sugars via SPPS have been described and thus could be incorporated into the synthesis of [Ser27, Nle70]hC5a and [Ser27, Nle70]hC5a-desArg to generate synthetic glycosylated hC5a to further explore differential binding, signaling, and activity.32

Interestingly, mC5a was difficult to observe using ESI-MS prior to disulfide formation. The main observable ion fragments were the six C-terminal residues (PVQLGR) and seven C-terminal residues (KPVQLGR) (669.5 and 797.7 Da, respectively), while the peaks corresponding to the full peptide were almost undetectable. Once mC5a was folded, the MS peaks corresponding to these small fragments disappeared, and signals corresponding to the full-length peptide were observed (Figure 1D). C-terminal ion fragments were also observed in ESI-MS for human C5a analogues; however, fragments corresponding to the full-length peptide were easily visualized from crude samples (Figure S2).

[Nle70]hC5a, [Ser27, Nle70]hC5a, [Ser27, Nle70]hC5a-desArg, and mC5a were structurally characterized using 1H NMR spectroscopy to assess residue dispersion and provide further validation of successful folding (Figure S6). Secondary Hα shifts were calculated for [Nle70]hC5a and compared with previously published secondary Hα shifts of recombinant hC5a.33 The closely matching secondary Hα shifts indicated that the synthetic [Nle70]hC5a produced in this work has the same structure as rhC5a and therefore the same disulfide connectivity (Figure S7).

Confirmation of In Vitro Activity at C5aR1

The ability of each analogue to induce ERK1/2 phosphorylation, a key signaling pathway employed by C5aR1, was assessed (Table S3). Both [Nle70]hC5a and [Ser27, Nle70]hC5a exhibited a similar pERK1/2 activity compared to rhC5a in CHO cells stably expressing C5aR1, eliciting full agonist responses and comparable potencies (rhC5a EC50 = 68.8 pM, [Nle70]hC5a EC50 = 44.4 pM, [Ser27, Nle70]hC5a EC50 = 39.6 pM) (Figure 2A,B).

Figure 2.

Figure 2

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Synthetic hC5a and mC5a show analogues pERK1/2 activity compared to recombinant C5a. (A) ERK1/2 phosphorylation of rhC5a (EC50 = 68.8 pM) vs [Nle70]hC5a (EC50 = 44.4 pM) normalized to rhC5a on CHO cells stably expressing C5aR1. rhC5a n = 6, [Nle70]hC5a n = 3. Error is ±SEM. (B) ERK1/2 phosphorylation induced by rhC5a (EC50 = 68.8 pM) vs [Ser27, Nle70]hC5a (EC50 = 39.6 pM) normalized to rhC5a on CHO cells stably expressing C5aR1. rhC5a n = 6, [Ser27, Nle70]hC5a n = 6. Error is ±SEM. (C) ERK1/2 phosphorylation of [Ser27, Nle70]hC5a (EC50 = 39.6 pM) vs [Ser27, Nle70]hC5a-desArg (EC50 = 170.8 pM) normalized to rhC5a on CHO cells stably expressing C5aR1. [Ser27, Nle70]hC5a n = 6, [Ser27, Nle70]hC5a -desArg n = 3. Error is ±SEM. (D) ERK1/2 phosphorylation of recombinant hC5a-desArg (EC50 = 274.3 pM) vs [Ser27, Nle70]hC5a-desArg (EC50 = 214.4 pM) normalized to recombinant hC5a-desArg on HMDMs. Recombinant hC5a-desArg n = 2, [Ser27, Nle70]hC5a-desArg n = 2. Error is ±SEM. (E) ERK1/2 phosphorylation when treated with increasing concentrations of PMX205 with a single concentration of [Ser27, Nle70]hC5a (1 nM) normalized to [Ser27, Nle70]hC5a without antagonist on CHO cells stably expressing C5aR1. IC50 of PMX205 inhibition of [Ser27, Nle70]hC5a is 96.7 nM. n = 3. Error is ±SEM. (F) ERK1/2 phosphorylation of rhC5a (EC50 = 294.7 pM) vs mC5a (EC50 = 213.3 pM) normalized to rhC5a on RAW264.7 cells. rhC5a n = 3, mC5a n = 3. Error is ±SEM.

[Ser27, Nle70]hC5a and [Ser27, Nle70]hC5a-desArg also elicited dose-dependent β-arrestin recruitment to C5aR2 similar to rhC5a (Figure S8).34 As expected, [Ser27, Nle70]hC5a-desArg showed reduced potency (170.8 pM) at C5aR1 compared to rhC5a (Figure 2C). The activity of [Ser27, Nle70]hC5a-desArg was also compared to rhC5a-desArg in human monocyte-derived macrophages (HMDMs) and demonstrated similar potency (rhC5a-desArg EC50 = 274.3 pM, [Ser27, Nle70]hC5a-desArg EC50 = 214.4 pM) (Figure 2D). Both rhC5a-desArg and [Ser27, Nle70]hC5a-desArg displayed a reduction in pERK1/2 signal at concentrations above 10 nM. A similar trend has previously been reported for rhC5a in primary human macrophages, most likely due to a C5aR2-induced reduction in C5aR1 signaling.35,36 To confirm the synthetic analogues of hC5a selectively activated hC5aR1, we pre-incubated C5aR1-CHO cells with increasing concentrations of the C5aR1 selective antagonist, PMX205, before treatment with 1 nM [Ser27, Nle70]hC5a (Figure 2E). PMX205 at a concentration of 10 or 100 μM completely ablated [Ser27, Nle70]hC5a-induced pERK1/2 response, and an IC50 of 96.7 nM for PMX205 was calculated, which is comparable to previous literature.37 Specificity of [Ser27, Nle70]hC5a was confirmed using HMDM’s, as treatment with 10 μM PMX205 ablated all pERK1/2 signaling elicited by 1 nM treatment of [Ser27, Nle70]hC5a (Figure S9). The assessment of mC5a activity was conducted in RAW264.7 cells, a mouse macrophage cell line. mC5a demonstrated comparable potency to rhC5a, although it displayed slightly higher efficacy at 10 nM (rhC5a EC50 = 294.7 pM, mC5a EC50 = 213.3 pM) (Figure 2F).

Generation and Validation of a Europium-Labeled hC5a Analogue

High-throughput binding assays that interrogate C5aR1/C5aR2-C5a interactions are crucial for the development of both agonists and antagonists at both receptors. The attachment of the DTPA chelating cage to the N-terminus of C5a allows for the incorporation of the lanthanide europium and subsequent functionalization of this peptide for C5aR1 and C5aR2 binding assays. Eu-DTPA-[Nle70]hC5a and Eu-DTPA-[Ser27, Nle70]hC5a were synthesized by our described method, and europium chelation was validated using ESI-MS (Table 1 and Figure 3A). The pERK1/2 activity of both europium-labeled hC5a analogues were assessed in CHO cells stably expressing C5aR1 whereby both analogues demonstrated analogous potency and efficacy to rhC5a (rhC5a EC50 = 68.8 pM, Eu-DTPA-[Nle70]hC5a EC50 = 88.6 pM, Eu-DTPA-[Ser27, Nle70]hC5a EC50 = 76.9 pM) (Figure 3B).

Figure 3.

Figure 3

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Generation and validation of europium-labeled hC5a. (A) DTPA-tetra (t-Bu ester) was used to attach the chelating cage to hC5a analogues [Nle70]hC5a and [Ser27, Nle70]hC5a during synthesis. Europium was captured and the labeled peptide was separated from free europium using an SPE cartridge. Correct product was confirmed using ESI-MS. (B) ERK1/2 phosphorylation of rhC5a (EC50 = 68.8 pM) vs Eu-DTPA-[Nle70]hC5a (EC50 = 88.6 pM) vs Eu-DTPA-[Ser27, Nle70]hC5a (EC50 = 76.9 pM) normalized to rhC5a on CHO cells stably expressing C5aR1. n = 3. Error is ±SEM. (C) Saturation binding assay performed on CHO cells stably expressing C5aR1. Nonspecific binding was assessed via co-incubation of Eu-DTPA-[Nle70]hC5a with unlabeled [Ser27, Nle70]hC5a (5 μM). Dissociation constant (Kd) is denoted by dotted line (14.3 ± 2.7 nM). n = 3. Error is ±SEM. (D) Saturation binding assay was performed on CHO cells stably expressing C5aR1. Nonspecific binding was assessed via co-incubation of Eu-DTPA-[Ser27, Nle70]hC5a with unlabeled [Ser27, Nle70]hC5a (5 μM). Dissociation constant (Kd) is denoted by dotted line (18.9 ± 5.9 nM). n = 3. Error is ±SEM. (E) Competition binding assay was performed on CHO cells stably expressing C5aR1. A single concentration of Eu-DTPA-[Ser27, Nle70]hC5a (10 nM) was incubated with increasing concentrations of unlabeled [Ser27, Nle70]hC5a (Ki = 11.6 nM) or PMX205 (Ki = 19.3 nM). n = 3. Error is ±SEM.

Saturation binding of both Eu-DTPA-[Nle70]hC5a and Eu-DTPA-[Ser27, Nle70]hC5a were conducted on CHO cells stably expressing C5aR1. The Kd values for Eu-DTPA-[Nle70]hC5a and Eu-DTPA-[Ser27, Nle70]hC5a were 14.3 and 18.9 nM, respectively. The calculated dissociation constants were not significantly different from each other (P = 0.4594; Figure 3B,C), suggesting that Cys27 is not significantly involved in the binding of hC5a to C5aR1.15 Competition binding of unlabeled [Ser27, Nle70]hC5a and PMX205 was then conducted to validate Eu-DTPA-[Ser27, Nle70]hC5a as a screening tool. Both [Ser27, Nle70]hC5a and PMX205 fully outcompeted Eu-DTPA-[Ser27, Nle70]hC5a at high concentrations (1 and 10 μM) with Ki’s of 11.6 and 19.3 nM, respectively. The development of a lanthanide-based binding assay for C5aR1 could therefore facilitate high-throughput screening of ligand binding and provides a useful tool in the discovery of next-generation agonists and antagonists for C5aR1, and potentially C5aR2.

Synthetic C5a and Recombinant C5a are Functionally Analogous In Vivo and Ex Vivo

Although our in vitro data suggests synthetically produced C5a and recombinant C5a are comparable, we sought to validate this further in both in vivo and ex vivo systems. As a measure of in vivo functionality, synthetic mC5a and rmC5a were administered intravenously via tail vein injection (50 μg/kg) to mice, followed by assessment of polymorphonuclear neutrophil (PMN) mobilization. Both synthetic and recombinant mC5a induced PMN mobilization compared to vehicle injection (P = <0.0001; Figure 4A), and the percentage of mobilized cells at 60 min after injection was not significantly different between the mC5a variants (P = 0.32; Figure 4A). The ability of [Ser27, Nle70]hC5a to elicit migration (chemotaxis) of neutrophils from human blood was also assessed and compared to rhC5a. Both [Ser27, Nle70]hC5a and rhC5a induced similar levels of neutrophil migration for all donors compared to vehicle (Figure 4B).

Figure 4.

Figure 4

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Synthetic mouse and human C5a are functionally active in vivo and ex vivo. (A) Time-course of mC5a-induced polymorphonuclear neutrophils (PMNs) in peripheral blood following tail vein injection. Data represented as a percentage of PMNs detected out of 400 white blood cells (WBCs). rmC5a and synthetic mC5a administered i.v. at a dosage of 50 μg/kg. n = 4. Error bars are SEM. (B) C5a-induced neutrophil migration from human blood (ex vivo). Relative migration (normalized to each donor) is shown. Data is n = 3 (3 different donors). Error bars are SEM.

Conclusions

In summary, this work describes the first efficient synthesis, folding, and purification of [Nle70]hC5a without the need for ligation chemistry. The outlined approach was also used to synthesize the analogue [Ser27, Nle70]hC5a and major metabolite [Ser27, Nle70]hC5a-des-Arg. In addition, we have demonstrated that this method can also be utilized to synthesize mC5a. All peptides produced were then compared at their respective species receptor (hC5aR1 for hC5a, mC5aR1 for mC5a), and the analogous potency to rhC5a in terms of pERK1/2 activity was confirmed. This study confirms that the cysteinylation at position 27 is not critical for pERK1/2 activity or neutrophil mobilization. We also confirm that the truncation of the C-terminal arginine of [Ser27, Nle70]hC5a leads to a reduction in pERK1/2 potency and efficacy at hC5aR1. hC5a was subsequently functionalized with Eu-DTPA for interrogation of C5aR1 binding. Both Eu-DTPA-[Nle70]hC5a and Eu-DTPA-[Ser27, Nle70]hC5a were produced and did not display a significant difference in their calculated Kd’s, suggesting that Cys27 is not significantly involved in hC5a binding to hC5aR1. Both mC5a and [Ser27, Nle70]hC5a were then validated to be functionally active in in vivo or ex vivo systems, respectively, as they both elicited neutrophil migration comparable to their recombinant counterparts. The synthetic approach outlined in this study serves as an alternative method of obtaining C5a for research purposes and allows for the easily integrated functionalization of hC5a for high-throughput receptor binding.

Methods

Peptide Synthesis and Purification

Peptides were synthesized on a CS Bio Co. CS336X peptide synthesizer using Fmoc (9-fluorenylmethyloxycarbonyl)-based solid-phase peptide synthesis (SPPS) on low-loaded 2-chlorotrityl chloride-linked resin 100–200 mesh 1% DVB (0.2 mmol/g; Mimotopes Pty Ltd.) at a 0.125 mmol scale. Briefly, 20% piperidine (Merck, Germany) was used to remove Fmoc for elongation of the peptide chain (2 × 5 min). A ratio of 4:4:4 equivalents of AA/HBTU/DIPEA was used for each coupling. At residue 20, equivalents were doubled (8:8:8 AA/HBTU/DIPEA), and each residue was double-coupled (2 × 30 min). Fmoc-Gly(Dmb)-OH was used for Gly-25, and the following aspartic acid residue (Asp-24) was triple-coupled. Diethylenetriamine-N,N,N″,N″-tetra-tert-butyl acetate-N′-acetic acid (Macrocyclics) was used to attach the DTPA chelating cage and was coupled manually at 4:4:8 equiv of AA/HBTU/DIPEA. Following the synthesis, peptides were cleaved from the resin, and side chain protecting groups were removed by treatment with TFA/TIPS/H2O/DODT at a ratio of 92.5:2.5:2.5:2.5 for 2 h at room temperature. Peptides were then precipitated with diethyl ether (Merck, Germany), filtered, and re-solubilized in 50/50 buffer A/B (45% ACN and 0.05% TFA in H2O) before being lyophilized. Crude peptides were then purified using reverse phase high performance liquid chromatography (RP-HPLC) with an increasing gradient of 1% buffer B (90% ACN and 0.045% TFA in H2O) in buffer A (0.05% TFA in H2O) per minute over 80 min (Phenomenex Jupiter 300 Å, 10 μm, 250 × 21.2 mm). HPLC fractions were analyzed by electrospray mass spectrometry (ESI-MS) (AB SCIEX API 2000), and fractions containing the desired product were combined.

Disulfide bonds were formed in oxidation buffer (0.1 M ammonium bicarbonate; pH 8) at a peptide concentration of 0.2 mg/mL over 48 h. Peptides were dissolved in minimal 50/50 buffer A/B (∼5 mg/mL; 45% ACN and 0.05% TFA in H2O) before being added dropwise to the oxidation buffer. For the oxidation of [Nle70]hC5a, 0.3 mM cysteine and 3 mM cystine were added to the oxidation buffer to facilitate the cysteinylation of Cys27. 1 M HCl (1 mL per 10 mL of oxidation buffer) was used to quench oxidation reactions prior to purification. Peptides were then purified by RP-HPLC until purity was >95%. Purity was assessed using analytical RP-HPLC (Agilent, 300 Å, 5 μm, 150 × 2.1 mm). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was performed to confirm correct mass to charge ratios (Bruker Autoflex).

Following disulfide bond formation, europium was chelated to DTPA-labeled analogues in 0.1 M ammonium acetate (pH 8) overnight at a concentration of 200 μM peptide and 600 μM EuCl3. The peptide was separated from salt using Sephadex G-25 resin and eluted in water. ESI-MS was used to confirm the chelation of europium.

Nuclear Magnetic Resonance (NMR) Spectroscopy

All peptides were prepared for NMR analysis by dissolving 1 mg of peptide in 550 μL of H2O/D2O (90:10), at pH 2.3, which is consistent with the conditions used previously to acquire NMR data for hC5a33. All spectra were recorded on a 700 MHz Bruker Avance III spectrometer equipped with a cryoprobe. 1D 1H NMR spectra were recorded at 298K. Two-Dimensional 1H–1H TOCSY and NOESY experiments were recorded, with mixing times of 80 and 200 ms, respectively, at 25 °C. The data were processed using Topspin 4.0.3 (Bruker), with the spectra being referenced to the solvent signal at 4.77 ppm. Spectra were assigned using sequential assignment strategies within the program CARA.38−40 Secondary structure was identified via the calculation of secondary Hα shifts using random coil values.41

In Vitro Activity Assays

Cell Culture

Chinese hamster ovary cells stably expressing the human C5aR1 (CHO-C5aR1) or human C3aR (CHO-C3aR) were maintained in Ham’s F-12 medium (Gibco) containing 10% fetal bovine serum (FBS) (Life Technologies), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 400 μg/mL G418 (Invivogen). Human embryonic kidney-293 (HEK293) cells were maintained in DMEM medium (Gibco) containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were maintained in T175 flasks (37 °C, 5% CO2) and subcultured at 80–90% confluency using 0.05% trypsin-EDTA (Gibco) in DPBS (LONZA). To ensure the consistency of cell function, cell morphology was continually monitored, and no cell lines were used beyond passage 20.

Human monocyte-derived macrophages (HMDM) were cultured as previously described.42 Human buffy coat blood from anonymous healthy donors was obtained through the Australian Red Cross Blood Service (Brisbane, Australia). CD14+ monocytes were isolated from blood using Lymphoprep density centrifugation (STEMCELL), followed by CD14+ MACS magnetic bead separation (Miltenyi Biotec). The isolated monocytes were differentiated for 7 days in IMDM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 15 ng/mL recombinant human macrophage colony-stimulating factor (hM-CSF) (Peprotech) on 10 mm square dishes (Sterilin). Nonadherent cells were removed by washing with DPBS, and the adherent-differentiated HMDMs were harvested by gentle scraping.

Human peripheral blood neutrophils (PMNs) were obtained from venous whole blood following the previously described protocol.27 Samples were collected from healthy volunteers under informed consent using venepuncture into BD K2EDTA Vacutainer blood collection tubes. Upon Lymphoprep (STEMCELL, Melbourne, Australia) density centrifugation (800g, 30 min, 22 °C), the cell pellet in the densest layer of the gradient containing a mixture of PMN and erythrocytes was collected. The residual erythrocytes were removed using hypotonic lysis. Isolated neutrophils were counted using a hemocytometer, and cell viability was ≥ 92% as assessed by Trypan blue exclusion.

pERK1/2 Signaling Assay

Ligand-induced phospho-ERK1/2 signaling was assessed using the AlphaLISA SureFire Ultra pERK1/2 (Thr202/Tyr204) kit (PerkinElmer) according to manufacturer’s instructions. Briefly, CHO-C5aR1, CHO-C3aR, or HMDMs were seeded (50 000/well) in tissue culture-treated 96-well plates (Corning) for 24 h and serum-starved overnight. All ligand dilutions were prepared in a serum-free medium (SFM) containing 0.1% BSA. For stimulation, cells were incubated with respective ligands for 10 min at room temperature and then immediately lysed using AlphaLISA lysis buffer on a microplate shaker (450 rpm, 10 min). For the detection of phospho-ERK1/2 content, cell lysate (5 μL/well) was transferred to a 384-well ProxiPlate (PerkinElmer) and added to the donor and acceptor reaction mix (2.5 μL/well, respectively) with 2 h incubation at room temperature in the dark. The plate was read using Tecan Spark 20M, following standard AlphaLISA settings.

BRET Assays Measuring β-Arrestin 2 Recruitment to C5a Receptors

The C5a-mediated β-arrestin 2 recruitment to C5aR1 and C5aR2, respectively, was measured using bioluminescence resonance energy transfer (BRET) as previously described.42 Briefly, HEK293 cells were transiently transfected with C5aR1-Renilla luciferase 8 (Rluc8) and β-arrestin 2-Venus or C5aR2-Venus and β-arrestin 2-Rluc8 constructs using XTG9 (Roche). At 24 h post-transfection, cells were gently detached using 0.05% trypsin-EDTA and seeded (100 000/well) onto white 96-well tissue culture plates (Corning) in phenol-red free DMEM containing 5% FBS. On the following day, cells were incubated with the substrate EnduRen (30 μM, Promega) for 2 h (37 °C, 5% CO2). On a Tecan Spark 20M microplate reader (37 °C), the BRET light emissions (460–485 and 520–545 nm) were continuously monitored for 25 reads with respective ligands added after the first 5 reads. The ligand-induced BRET ratio was calculated by subtracting the Venus (520–545 nm) over Rluc8 (460–485 nm) emission ratio of the vehicle-treated wells from that of the ligand-treated wells.

Saturation Binding Assay

CHO-C5aR1 cells were seeded into poly-lysine coated white-walled 96-well flat-bottom plates at 50 000 cells/well in complete Ham’s F-12 media. Eu-DTPA-ligand dilutions were made up in binding buffer (0.5% BSA, 50 mM Tris-HCl pH 7.4, 0.9% NaCl, and 20 μM EDTA) from 100 to 1.56 nM. An identical dilution series was also made up in buffer containing 5 μM concentration of the unlabeled ligand to assess nonspecific binding. Cells were washed twice with wash buffer (15 mM Tris-Cl pH 7.8 and 0.9% NaCl) before the addition of 100 μL drug dilutions. Plates were incubated on ice (4 °C) for 1 h. Following incubation, the binding buffer was aspirated off, wells were washed twice with binding buffer, and 100 μL of DELFIA enhancement solution was added to each well. The plate was shaken on an orbital shaker at 300 rpm for 15 min before having time-resolved fluorescence read on the Tecan Spark 20M plate reader as per manufacturer’s instructions.

Competition Binding Assay

CHO-C5aR1 cells were seeded into poly-lysine-coated white-walled 96-well flat-bottom plates at 50 000 cells/well in complete Ham’s F-12 media. Ligand dilutions were made up in binding buffer (0.5% BSA, 50 mM Tris-HCl pH 7.4, 0.9% NaCl, 20 μM EDTA, and 10 nM Eu-DTPA-[Ser27, Nle70]hC5a) from 10 μM to 10 pM. Cells were washed twice with wash buffer (15 mM Tris-Cl pH 7.8 and 0.9% NaCl) before the addition of 100 μL drug dilutions. Plates were incubated on ice (4 °C) for 1 h. Following incubation, the binding buffer was aspirated off, wells were washed twice with binding buffer, and 100 μL of DELFIA enhancement solution was added to each well. The plate was shaken on an orbital shaker at 300 rpm for 15 min before having time-resolved fluorescence read on the Tecan Spark 20M plate reader as per manufacturer’s instructions.

Data Collection, Processing, and Analysis

All experiments were conducted in triplicate and repeated on at least 3 separate days (for cell lines) or using cells from at least 3 donors (for HMDMs) unless otherwise specified. The data was analyzed using GraphPad software (Prism 9.0) and expressed as mean ± standard error of the mean (SEM). The data from each repeat was normalized accordingly before being combined. For all dose–response assays, logarithmic concentration–response curves were plotted using combined data and analyzed to determine the respective potency values.

Functional Assays

In Vivo Mouse Neutrophil Mobilization Assay

C57BL/6J mice (n = 4) were administered with rhC5a or synthetic mC5a at a dose of 50 μg/kg via intravenous injection (tail vein). After injection, one drop of blood was collected from the tail tip to make a blood smear on a slide at 0, 15, 30, and 60 min. Blood smears were stained using microscopy Hemacolor Rapid staining of blood smear kit (Merck, Germany). Briefly, blood smears were fixed in Hemacolor Solution 1 (methanol). The slides were then stained with Hemacolor Solution 2 (Eosin Y), followed by Hemacolor Solution 3 (Azur B). The slides were washed with 1× PBS (pH 7.2) and mounted with dibutyphthalate polystyrene xylene. Using 20×/0.4 NA objective on an Olympus CX21 microscope, the first 400 white blood cells were counted, and the proportion of neutrophil (granules that are light violet) was calculated.

C5a-induced PMN migration was assessed using 6.5 mm Transwell polycarbonate membrane inserts with 3.0 μm pore (Corning, New York) to create a modified Boyden chamber. Freshly isolated PMNs were resuspended in a HBSS-based migration buffer (containing calcium and magnesium, supplemented with 20 mM HEPES and 0.5% BSA) and seeded onto inserts (600 000/well). To initiate cell migration, [Ser27, Nle70]hC5a or rhC5a (1 nM) prepared in migration buffer was added to the receiver wells in duplicates. After 60 min migration (37 °C, 5% CO2), the unmigrated cells on the upper side of the membrane were removed using a cotton swab. Migrated cells were detached by adding 500 μL/well Accumax solution (Thermo Fisher Scientific, Melbourne, Australia) to the receiver wells (10 min, room temperature) and then counted using a hemocytometer.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00199.

  • Synthesis yields of human C5a synthesis; listed peptide molecular weights before and after disulfide bond formation; ERK1/2 activity/potency for human and mouse C5a analogues in CHO-hC5aR1 cells or RAW264.7 cells, respectively; analytical HPLC and mass spectra of crude C5a analogues; analytical HPLC and mass spectra of pure linear and oxidized C5a analogues; time-course of [Ser27, Nle70]hC5a disulfide formation over 48 h; 1D 1H NMR spectra of C5a analogues; secondary Hα shifts of synthetic and recombinant hC5a; activity/potency of β-arrestin recruitment of [Ser27, Nle70]hC5a and [Ser27, Nle70]hC5a-desArg at C5aR2; and ERK1/2 activity of [Ser27, Nle70]hC5a with or without PMX205 in HMDM’s (PDF)

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

T.M.W., R.J.C., and D.M.G. contributed to the study conception and design. Material preparation, data collection, and analysis were performed by D.M.G., X.X.L., C.D.P., C.S.C., and J.D.L. D.M.G. synthesized peptide compounds. D.M.G. and X.X.L. performed in vitro assays. C.D.P. and K.J.R. performed NMR spectroscopy. J.D.L. and C.S.C. performed in vivo assays. D.M.G. and R.J.C. wrote the manuscript, and all authors contributed to revisions of the manuscript.

This work was supported by the National Health and Medical Research Council of Australia (NHMRC) [Grant APP1118881].

The authors declare no competing financial interest.

Supplementary Material

pt1c00199_si_001.pdf (1.7MB, pdf)

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