Mitocryptide-2: Identification of Its Minimum Structure for Specific Activation of FPR2–Possible Receptor Switching from FPR2 to FPR1 by Its Physiological C-terminal Cleavages

Mitocryptides are a novel family of endogenous neutrophil-activating peptides originating from various mitochondrial proteins. Mitocryptide-2 (MCT-2) is one of such neutrophil-activating peptides, and is produced as an N-formylated pentadecapeptide from mitochondrial cytochrome b. Although MCT-2 is a specific endogenous ligand for formyl peptide receptor 2 (FPR2), the chemical structure within MCT-2 that is responsible for FPR2 activation is still obscure. Here, we demonstrate that the N-terminal heptapeptide structure of MCT-2 with an N-formyl group is the minimum structure that specifically activates FPR2. Moreover, the receptor molecule for MCT-2 is suggested to be shifted from FPR2 to its homolog formyl peptide receptor 1 (FPR1) by the physiological cleavages of its C-terminus. Indeed, N-terminal derivatives of MCT-2 with seven amino acid residues or longer caused an increase of intracellular free Ca2+ concentration in HEK-293 cells expressing FPR2, but not in those expressing FPR1. Those MCT-2 derivatives also induced β-hexosaminidase secretion in neutrophilic/granulocytic differentiated HL-60 cells via FPR2 activation. In contrast, MCT-2(1–4), an N-terminal tetrapeptide of MCT-2, specifically activated FPR1 to promote those functions. Moreover, MCT-2 was degraded in serum to produce MCT-2(1–4) over time. These findings suggest that MCT-2 is a novel critical factor that not only initiates innate immunity via the specific activation of FPR2, but also promotes delayed responses by the activation of FPR1, which may include resolution and tissue regeneration. The present results also strongly support the necessity of considering the exact chemical structures of activating factors for the investigation of innate immune responses.


Introduction
Neutrophils are a type of leukocyte that are involved in the innate defense system [1][2][3]. Neutrophils comprise the majority of peripheral leukocytes and normally exist in the bloodstream to monitor for infection and tissue damage. When tissue injury occurs due to bacterial infections or internal tissue damage, neutrophils immediately migrate to and infiltrate the injury site. The infiltrated neutrophils are then activated and exert their functions, including superoxide production and phagocytosis of invading bacterial components and toxic substances.
Bacterial N-formylated proteins and peptides, including formyl-Met-Leu-Phe (fMLF) [4,5], complement related factors such as component 5a [6,7], and some chemokines such as IL-8 [8,9] chemoattract and activate neutrophils to promote inflammatory reactions. Moreover, various mitochondrial-derived peptides that also activate neutrophils were recently In this study, we investigated the structure-activity relationships of MCT-2 and its derivatives to elucidate how FPR2 recognizes MCT-2. We also explored the time-dependent alterations of the molecular forms of MCT-2 in serum and attempted to elucidate the meaning of these alterations in innate immune responses.  and Its Derivatives on [Ca 2+ ] i in HEK-293 Cells Stably Expressing FPR1 or FPR2

Effects of Substituting Ala for Each Amino Acid Residue of MCT-2(1-15) on β-Hexosaminidase Release by Differentiated HL-60 Cells
We examined the effect of substituting Ala for each amino acid residue in MCT-2(1-15) on the stimulation of β-hexosaminidase release to elucidate the contribution of each side chain structure to this process. As the MCT-2(1-7) structure with an N-formyl group was indicated as crucial for the activation of FPR2 (Figures 1-3), we investigated the effect of substituting Ala for each amino acid residue in positions one to seven within MCT-2(1-15). The replacement of Met 1 of MCT-2(1-15) with Ala caused a 30% reduction of the maximum response with a dramatic (>500-fold) increase in the EC 50 value compared with MCT-2(1-15) ( Figure 4A and Table 2), indicating that the Met 1 side chain is important for not only the affinity of MCT-2(1-15) to FPR2, but also for FPR2 activation. The substitution of Met 4 , Arg 5 , Lys 6 , or Ile 7 with Ala also caused an increase in the EC 50 value with the same level of maximum response as MCT-2 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In contrast, the replacement of Thr 2 with Ala promoted a remarkable decrease in the EC 50 value without affecting the maximum response, although the substitution of Pro 3 with Ala had no effect on the EC 50 value. These results suggest that the side chains of Met 4 , Arg 5 , Lys 6 , and Ile 7 , and Thr 2 within MCT-2(1-15) contribute positively and negatively, respectively, to its affinity to bind to FPR2. not only the affinity of MCT-2(1-15) to FPR2, but also for FPR2 activation. The substitution of Met 4 , Arg 5 , Lys 6 , or Ile 7 with Ala also caused an increase in the EC50 value with the same level of maximum response as MCT-2 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In contrast, the replacement of Thr 2 with Ala promoted a remarkable decrease in the EC50 value without affecting the maximum response, although the substitution of Pro 3 with Ala had no effect on the EC50 value. These results suggest that the side chains of Met 4 , Arg 5 , Lys 6 , and Ile 7 , and Thr 2 within MCT-2(1-15) contribute positively and negatively, respectively, to its affinity to bind to FPR2.  The differentiated HL-60 cells were stimulated by MCT-2(1-15) or its derivatives at 37 • C for 10 min, and the amount of the released β-hexosaminidase was quantified as described in "Materials and Methods". The ability of each peptide to cause β-hexosaminidase release is expressed as a percentage of enzyme secretion promoted by 10 µM MCT-2(1-15). Data are expressed as the mean ± SE of four to six independent experiments. Table 2. Amino acid sequences of MCT-2(1-15) and its Ala-substituted derivatives and their EC 50 values and maximum effects on the induction of β-hexosaminidase release from differentiated HL-60 cells.

Peptide
Sequence EC 50  We also examined the effect of substituting Ala for each amino acid residue in the MCT-2(8-15) sequence within MCT-2(1-15) because this structure may be important for the binding affinity between MCT-2(1-15) and FPR2, but not the receptor activation described above. The substitution of Leu 10 , Leu 13 , or Ile 14 with Ala caused an increase in the EC 50 value with the same level of maximum response as MCT-2(1-15) ( Figure 4B and Table 2). In contrast, the replacement of Pro 9 with Ala promoted a remarkable decrease in the EC 50 value without affecting the maximum response [EC 50 : 3 ± 2 nM, Figure 4B and Table 2], although the substitution of Asn 8 , Met 11 , Lys 12 , or Asn 15 with Ala had no effect. These results suggest that the Leu 10 , Leu 13 , or Ile 14 , and Pro 9 side chains within MCT-2(1-15) contribute positively and negatively, respectively, to its affinity to bind to FPR2.

Circular Dichroic Spectra of MCT-2(1-15) and Its Derivatives
It was suggested that the Thr 2 , Met 4 , Arg 5 , Lys 6 , Ile 7 , Pro 9 , Leu 10 , Leu 13 , and Ile 14 side chains within MCT-2(1-15) had an effect on its binding affinity to FPR2 and that the MCT-2(8-15) structure was important for binding to FPR2. Thus, the secondary structures of MCT-2(1-15) and its derivatives were analyzed using circular dichroic (CD) spectra, which is an excellent tool for the rapid investigation of secondary structures. As we reported previously [45], the CD spectrum of MCT-2(1-15) exhibited two minima at 225 nm and 205 nm in TFE solution, suggesting that MCT-2(1-15) predominantly contained an α-helical structure in hydrophilic circumstance ( Figure 5A). Similarly, the spectra of the N-terminal MCT-2 derivatives that were truncated by one to six amino acid residues from the C-terminus of MCT-2(1-15) at 100 µM also showed two minima at 225 nm and 202-208 nm in TFE solution, although these minima were consecutively attenuated by the C-terminal truncations ( Figure 5B-G). Moreover, the spectra of MCT-2(1-8) and MCT-2(1-7) displayed a minimum at approximately 200 nm in TFE solution ( Figure 5H,I), proposing that MCT-2(1-8) and MCT-2(1-7) do not contain defined secondary structures, even under hydrophilic conditions. In contrast, the CD spectra of all of these derivatives including MCT-2(1-15) at 100 µM exhibited a minimum at approximately 200 nm in a hydrophilic phosphate buffer ( Figure 5), proposing that MCT-2(1-15) and its derivatives did not form defined secondary structures in hydrophilic conditions.
MCT-2(1-6) did not induce an increase of [Ca 2+ ] i in HEK-293 cells expressing either FPR2 or FPR1, even at 100 µM; nevertheless, it caused β-hexosaminidase secretion (Figure 2). The apparent discrepancy between the induction of β-hexosaminidase release and increase in [Ca 2+ ] i stimulated by MCT-2(1-6) may be a result of its weak stimulatory activity for not only FPR1, but also FPR2; the weak stimulation of β-hexosaminidase release by MCT-2(1-6) was presumably a consequence of the slight activation of both FPR1 and FPR2, although this was not evident in HEK-293 cells expressing FPR1 or FPR2. The idea that MCT-2(1-6) weakly activates FPR1 and FPR2 for the induction of β-hexosaminidase secretion was also supported by the use of selective inhibitors against FPR1 or FPR2, i.e., β-hexosaminidase release stimulated by MCT-2(1-6) was partially inhibited by either CysH or PBP10, and was completely prevented by a combination of both inhibitors ( Figure 3A-C).

Secondary Structures of MCT-2(1-15) and Its Derivatives for the Interaction with FPR2
In the present study, we showed that the MCT-2(1-7) structure within MCT-2(1-15) is required to induce the maximum response by FPR2 activation. What is the role of the C-terminal MCT-2(8-15) sequence within MCT-2(1-15)? Truncation of one to eight amino acid residues from the C-terminus of MCT-2(1-15) had no effect on the maximum response for the stimulation of β-hexosaminidase release, but did cause a consecutive increase of EC 50 values ( Figure 2B and Table 1), suggesting that the MCT-2(8-15) structure contributes to the binding affinity of MCT-2(1-15) to FPR2, but is not essential for the receptor activation itself. Especially, the removal of the Ile 14 , Leu 13 , Met 11 , and Leu 10 side chains from MCT-2(1-15) significantly increased the EC 50 values, suggesting that these hydrophobic amino acid residues are important for the affinity of MCT-2(1-15) with FPR2. Many bioactive peptides form amphipathic α-helical structures when interacting with the cell membrane, and the hydrophobic side chains of those peptides influence their affinity for the cell membrane and their receptors [52,53]. Indeed, MCT-2(1-15) exhibited α-helical signals in CD spectra in hydrophilic conditions, and the truncation of one to eight amino acid residues from its C-terminus caused a simultaneous decrease of the α-helical signals ( Figure 5). These findings propose that the α-helical structure of MCT-2(1-15) formed in hydrophobic circumstance may also contribute to its interaction with FPR2. Here, the substitution of Pro 9 in MCT-2(1-15) with Ala remarkably decreased the EC 50 value for β-hexosaminidase release, i.e., its activity was potentiated by the exchange (Figure 4B). Taken together with our previous findings that the replacement of Pro 9 in MCT-2(1-15) with Ala increased the α-helical content in hydrophilic conditions [45], the importance of the amphiphilic α-helical structure of the C-terminal part of MCT-2(1-15) for the improvement of its affinity for FPR2 was also supported by these results.
In this study, the Thr 2 side chain was also suggested to contribute negatively to the affinity of MCT-2(1-15) for FPR2, that is, the replacement of Thr 2 in MCT-2(1-15) with Ala promoted a remarkable decrease in the EC 50 value (Table 2); nevertheless, this substitution did not influence the α-helical content of MCT-2(1-15) [45]. Hence, the increase in activity by its replacement may be a result of an increase in hydrophobicity at position two that improves its binding affinity with FPR2.

Binding Characteristics of MCT-2(1-15) and Its Derivatives to FPR1 and FPR2
Recently, the tertiary structure of the FPR2-G i complex analyzed by cryo-electron microscopy (EM) has been reported, and that of the FPR1-G i complex was also predicted based on the structure of FPR2 by Zhuang et al. [54]. In addition, formylated peptides containing fMLF were docked to those receptors. These findings suggested that the Nformyl groups of those peptides interacted with the Asp 106 , Arg 201 , and Arg 205 residues distributed at the bottom of the ligand-binding cavities of FPR1 and FPR2 for the activation of both receptors. Thus, we simulated the docking of MCT-2(1-15) and its derivatives to FPR2 and FPR1 using the Glide program in Schrödinger, which was the program used by Zhuang et al. [54] (Figure 7, Marutani et al., manuscript in preparation). In brief, the Nformyl group of MCT-2(1-15) and its N-terminal derivatives longer than seven amino acid residues were also shown to interact with Asp 106 , Arg 201 , and Arg 205 of FPR2 but not those of FPR1 because these peptides caused steric hindrance on binding to the cavity of FPR1 ( Figure 7A and C vs. Figure 7E,G). Moreover, the Met 1 side chains of those peptides filled the space at the bottom of the ligand-binding cavity of FPR2 ( Figure 7A,C), presumably contributing to the stabilization of the interaction between the N-formyl groups of those peptides and Asp 106 , Arg 201 , and Arg 205 of FPR2 for receptor activation. In contrast, MCT-2(1-4) just fit into the binding cavity of FPR1 to promote the interaction between its N-formyl group and Asp 106 , Arg 201 , and Arg 205 ( Figure 7F,H). However, the interaction between MCT-2(1-4) and the ligand-binding cavity of FPR2 was not stabilized due to the lack of hydrophobic and hydrogen bonding interactions as well as the large binding cavity ( Figure 7B  In contrast, MCT-2(1-4) just fit into the binding cavity of FPR1 to promote the interaction between its N-formyl group and Asp 106 , Arg 201 , and Arg 205 ( Figure 7F,H). However, the interaction between MCT-2(1-4) and the ligand-binding cavity of FPR2 was not stabilized due to the lack of hydrophobic and hydrogen bonding interactions as well as the In addition, the Arg 5 or Lys 6 residue of MCT-2(1-7) and the C-terminal carboxyl group of MCT-2(1-4) were exhibited to form hydrogen bonds with the Asp 281 residue of FPR2 and the Arg 84 residue of FPR1 ( Figure 7C,H), respectively, which are distributed at the top of the binding cavities of those receptors, proposing that these hydrogen bonding interactions are of importance for stabilizing further receptor-peptide binding. The present simulation results can well explain those from structure-activity studies of the peptides in the present study in which MCT-2(1-7) and its derivatives longer than seven amino acid residues specifically activated FPR2, whereas MCT-2(1-4) specifically activated FPR1.

Alteration of the Molecular Forms of MCT-2(1-15) in the Bloodstream
Since the receptor preference of MCT-2(1-15) and its derivatives critically depends on the length of its C-terminus, changes in the molecular forms of MCT-2(1-15) in serum were investigated. MCT-2(1-15) in serum was detected within 4 h after incubation, and its half-life was approximately 1 h ( Figure 6). In addition, the production of MCT-2-related FPR2 specific agonists MCT-2(1-11) and MCT-2(1-10) was observed at 1 h after incubation, but they were no longer present at 4 h, similar to MCT-2(1-15) ( Figure 6). Since MCT-2(1-15) is proposed to be released into the bloodstream from injury tissues as discussed below, these results suggest that MCT-2(1-15) released into the bloodstream initially activates FPR2 for several hours. In contrast, the MCT-2-related FPR1 specific agonist MCT-2(1-4) was found at low levels at 1 h after incubation; its levels then increased gradually over time, and its maximum amount was observed at 4 h. Moreover, MCT-2(1-4) was still present at 48 h ( Figure 6). These findings propose that MCT-2(1-15) released into the bloodstream is degraded and the resulting product, MCT-2(1-4), induces the activation of FPR1 following FPR2 activation and continues to activate FPR1 for over 48 h.

Possible Physiological Roles of the Receptor Preference Shift of MCT-2(1-15) from FPR2 to FPR1
The results of this study demonstrate that the receptor preference of MCT-2(1-15) is shifted from FPR2 to FPR1 by the cleavage of its C-terminus. What is the physiological significance of this shift in receptor preference? FPR1 and FPR2 play critical roles in inflammation including proinflammatory responses, subsequent resolution, and wound healing/tissue regeneration. Specifically, FPR1 and FPR2 are expressed mainly by inflammatory immune cells including neutrophils, monocytes, and monocyte-derived macrophage cells such as tissue-resident macrophages and microglia [35,37,38], and FPR2 is also expressed by a variety of cells including microvascular endothelial cells [38,39]. FPR1 and FPR2 have roles in the mechanisms concerning the infiltration of neutrophils and macrophages into injury sites, and their activation causes various inflammatory responses, including phagocytosis, superoxide generation, and inflammatory cytokine production [35,37,38]. In addition, there is evidence indicating that the activation of FPR2 increases the vascular permeability of endothelial cells [38,39], suggesting a further promotion of neutrophil infiltration from the bloodstream into injury sites following receptor activation in the initial stage of inflammation. Thus, the activation of FPR1 and FPR2 expressed by neutrophils, macrophages, and endothelial cells induces various innate immune responses initiated by the infiltration and activation of neutrophils. Oppositely, it is known that liganded FPR2 suppresses the production of inflammatory cytokines following the acute proinflammatory responses [38,[55][56][57][58]. Moreover, FPR1 activation has been demonstrated to promote wound healing/tissue regeneration, including cell proliferation [40][41][42][43][44].
MCT-2(1-15) is the only endogenous N-formylated peptide whose chemical structure has been determined so far, and the presence of MCT-2-related peptides in mtDAMPs was recently observed by immunoblot analysis using a monoclonal antibody against MCT-2 (Tsutsumi et al., unpublished observation). Taken together with these findings, the present results suggest the hypothesis that MCT-2(1-15) is initially released into the bloodstream from damaged cells following tissue injury, and then activates FPR2 specifically to induce acute innate immune responses, including the infiltration and activation of neutrophils ( Figure 8). MCT-2(1-15) is then degraded in damaged tissues as well as in the bloodstream over time, and the resulting MCT-2-related FPR1 specific agonist MCT-2(1-4) activates FPR1 to promote delayed responses, which may include resolution and wound healing/tissue regeneration. Indeed, we recently found that a specific neutralizing monoclonal antibody against MCT-2 attenuated the infiltration of neutrophils into injured liver tissue in acetaminophen-or LPS-induced inflammation and prolonged the survival of mice, suggesting that MCT-2(1-15) and its derivatives play a critical role in innate immunity (Takamuro et al., manuscript in preparation). The present findings also indicate the crucial importance of investigating the molecular forms and/or exact chemical structures of those activating factors to elucidate the mechanisms underlying innate immune responses. are thought to activate FPR1 and/or FPR2 to induce innate immune responses [15,17,28] MCT-2(1-15) is the only endogenous N-formylated peptide whose chemical structure has been determined so far, and the presence of MCT-2-related peptides in mtDAMPs was recently observed by immunoblot analysis using a monoclonal antibody against MCT-2 (Tsutsumi et al., unpublished observation). Taken together with these findings, the pre sent results suggest the hypothesis that MCT-2(1-15) is initially released into the blood stream from damaged cells following tissue injury, and then activates FPR2 specifically to induce acute innate immune responses, including the infiltration and activation of neu trophils (Figure 8). MCT-2(1-15) is then degraded in damaged tissues as well as in the bloodstream over time, and the resulting MCT-2-related FPR1 specific agonist MCT-2(1-4) activates FPR1 to promote delayed responses, which may include resolution and wound healing/tissue regeneration. Indeed, we recently found that a specific neutralizing monoclonal antibody against MCT-2 attenuated the infiltration of neutrophils into injured liver tissue in acetaminophen-or LPS-induced inflammation and prolonged the surviva of mice, suggesting that MCT-2(1-15) and its derivatives play a critical role in innate im munity (Takamuro et al., manuscript in preparation). The present findings also indicate the crucial importance of investigating the molecular forms and/or exact chemical struc tures of those activating factors to elucidate the mechanisms underlying innate immune responses.

Measurement of [Ca 2+ ] i
The increase in [Ca 2+ ] i stimulated by peptides was assessed, as described previously [11,36]. In brief, HEK-293 cells stably expressing FPR1 or FPR2 with a Gα 16 type of G protein were washed twice with a HEPES-Na solution (5 mM HEPES, 140 mM NaCl, 4 mM KCl, 1 mM NaH 2 PO 4 , 1 mM MgCl 2 , 1.25 mM CaCl 2 , 11 mM glucose, and 0.2% BSA, pH 7.4). The Ca 2+ -sensitive fluorescence reagent Fura-2-acetoxymethyl ester (Dojin, Kumamoto, Japan) was added to the cell suspension (4 mL; final concentration: 4 µM). The reaction mixture was shielded from light and shaken gently at 37 • C for 60 min to load the cells with Fura-2. The cells were subsequently washed twice with the HEPES-Na solution and a cell suspension was diluted to a final density of 5.0 × 10 5 cells/mL. The cell suspension (1 mL) was placed into a cuvette and stimulated by peptide solutions (5 µL) with stirring at 37 • C. The ratio of fluorescence intensity at 500 nm by excitation wavelengths of 340 nm and 380 nm was measured using a fluorometer (CAF-100; Japan Spectroscopic Co., Tokyo, Japan).
β-Hexosaminidase activity in the supernatant was measured as described previously [63]. Briefly, 90 µL supernatant was transferred to each well of a 96-well microtiter plate, and 60 µL of a substrate solution for β-hexosaminidase [10 mM p-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich), 40 mM citrate, and 70 mM NaHPO 4 , pH 4.5] was added to initiate the enzyme reaction. After incubation of the plate at 37 • C for 1 h, 100 µL of 400 mM glycine (pH 10.7) was added to stop the reaction. The absorbance at 415 nm for the resulting p-nitrophenol and at 490 nm for the reference was measured using a microtiter plate reader (Viento XS; BioTek Instruments, Winooski, VT, USA).
The ability of each peptide to induce β-hexosaminidase release was expressed as a percentage of enzyme secretion promoted by 10 µM MCT-2(1-15) that induced the maximum response for the elucidation of full or partial agonists to the activity of MCT-2(1-15) (Figures 2 and 4, Table 2) or a percentage of the total enzyme activity, which was the enzyme activity released after disruption of the cells with 0.05% Triton X-100 (Figure 3).

Measurement of Circular Dichroic Spectra
CD spectra of the synthetic peptides were obtained at 25 • C using a J-820 spectrometer (Jasco, Tokyo, Japan) in a quartz cell with a 0.1 cm path length. Spectra were collected between 190 nm and 250 nm with a scan speed of 50 nm/min, response time of 1 s, and bandwidth of 1 nm. Peptide samples with a final concentration of 100 µM were prepared in 10 mM phosphate buffer (pH 7.4) containing 0% or 50% TFE. The baseline scan, which was acquired by measuring the buffer alone, was subtracted from the experimental readings. CD data, which were collected every 1 nm, were the average of 5 scans. The results were expressed as the optical rotation (mdeg).

Analysis of Time-Dependent Alterations of MCT-2 Molecular Forms in Serum
The animal experiments were conducted under the guidance of the Animal Care and Use Committee of the Nagahama Institute of Bio-Science and Technology (Approved No. 047). C57BL/6JJcl mice were purchased from Clea Japan (Tokyo, Japan). All mice were maintained in the Animal Research Facility at the Nagahama Institute of Bio-Science and Technology.
Male C57BL/6JJcl mice, 12-14 weeks of age, weighing 25-30 g, were anesthetized (50 mg/kg pentobarbital), and blood was collected from the caudal vena cava and stored overnight at 4 • C. The blood sample was centrifuged at 4 • C and 20,000× g for 20 min, and the supernatant was transferred to a new tube as mouse serum and stored at −80 • C. MCT-2(1-15) was incubated in mouse serum at a final concentration of 500 µM at 37 • C, and aliquots (100 µL) were collected from the incubation mixture after 0, 1, 2, 4, 24, and 48 h. These aliquots were mixed with TCA (final concentration: 3% w/v) and kept on ice for 30 min to precipitate denatured proteins. The samples were centrifuged at 4 • C and 13,000× g for 15 min, and the supernatants were analyzed by RP-HPLC on a C 18 column (4.6 × 150 mm, Cosmosil; Nacalai Tesque, Inc.). RP-HPLC peaks that contained MCT-2(1-15) and its derivative peptides were analyzed by MALDI-TOF-MS to identify their molecular forms.

Statistical Analysis
Data are expressed as the mean ± standard error (SE) in experiments containing multiple data points. Statistical comparisons between two groups were performed using Student's t-test, and values of p < 0.01 were considered significant.

Conclusions
In the present study, we demonstrated that FPR2 recognizes the MCT-2(1-7) structure with an N-formyl group for its specific activation to induce neutrophilic functions. N-terminal MCT-2 derivatives shorter than seven amino acid residues were shown to lose their specificity for FPR2 and gain the ability to activate FPR1. Moreover, we showed that MCT-2(1-15) was degraded in serum over time, and the MCT-2-related FPR1 specific agonist MCT-2(1-4) was produced, suggesting that the receptor preference of MCT-2 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) in the bloodstream is shifted from FPR2 to FPR1 over time by the cleavage of its C-terminus by various proteases. Thus, MCT-2 is proposed to be a factor that controls not only the ini-tiation of innate immune responses against tissue injury, but also delayed responses via the activation of FPR1, which may relate to resolution and wound healing/tissue regeneration. In addition, because the docking simulation of MCT-2(1-15) and its derivatives to FPR1 or FPR2 well explained the receptor-specific activation mechanisms by those peptides as well as the results of structure-activity relationships, the present findings with structural information of FPR2 and FPR1 are expected to accelerate the development of specific antagonists for not only FPR2 but also FPR1 for the treatment of various inflammatory diseases including the recent epidemic of pneumonia that often causes multiple organ failure.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data and materials presented in this article and in the supplementary information are available from the corresponding author upon reasonable request.

Acknowledgments:
The authors are grateful to Yasushi Kawai, Osamu Saitoh, and Shintaro Nomura for their critical reading of the manuscript and helpful advice. We also thank Masafumi Shionyu for helpful discussions and suggestions related to the docking between MCT-2 or its derivatives and FPR2 or FPR1.

Conflicts of Interest:
The authors have no financial conflicts of interest to declare.