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.

Takayuki Marutani,Tomoyuki Miyaji,Kodai Nishino,Keisuke Kamada,Hidehito Mukai,Yoshiaki Kiso,Koji Ohura

doi:10.3390/ijms22084084

Abstract

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.

Highlights

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
To further elucidate the involvement of formyl peptide receptor 1 (FPR1) and formyl peptide receptor 2 (FPR2) in β-hexosaminidase release from differentiated HL-60 cells stimulated with MCT-2(1–15) and its derivatives, we examined the inhibitory effects of inhibitors against FPR1 [cyclosporin H (CysH)] and FPR2 (PBP10) on this process
FPR1, which recognizes N-formylated peptides, and its homolog FPR2 are expressed in neutrophils and neutrophilic differentiated HL-60 cells [33,34,35,38,48,49,50,51], and the endogenous pentadecapeptide MCT-2(1–15) activates FPR2 but not FPR1 [36,45]

Summary

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 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. Various mitochondrial-derived peptides that activate neutrophils were recently identified in mammalian tissues [10,11,12,13,14]. We found the possible presence of many mitocryptides that were derived from various mitochondrial proteins to induce the migration and activation of neutrophils [10,14]

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Discussion

Conclusion