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Mitocryptide-2: Purification, Identification, and Characterization of a Novel Cryptide That Activates Neutrophils ¹

Hidehito Mukai^2,*,,,, Tetsuo Seki^3,*, Hiroko Nakano^3,*, Yoshinori Hokari^*, Toshifumi Takao^¶, Masanori Kawanami, Hiroyuki Tsukagoshi^||, Hirokazu Kimura^#, Yoshiaki Kiso, Yasutsugu Shimonishi^4,¶, Yoshisuke Nishi^4, and Eisuke Munekata^*

^* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan; Laboratory of Peptide Biosignal Engineering, Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan; Laboratory of Life Science and Biomolecular Engineering, JT Inc., Yokohama, Kanagawa, Japan; 21st Century COE Program, Kyoto Pharmaceutical University, Yamashina, Kyoto, Japan; ^¶ Institute for Protein Research, Osaka University, Suita, Osaka, Japan; ^|| Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma, Japan; and ^# Infectious Diseases Surveillance Center, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide "mitocryptide-2" (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide. Synthetic MCT-2 and its human homolog hMCT-2 induced β-hexosaminidase release in and chemotaxis of HL-60 cells differentiated into neutrophilic/granulocytic cells. The induction of β-hexosaminidase release, chemotaxis, and the increase in the intracellular free Ca²⁺ concentration by hMCT-2 were completely suppressed by pertussis toxin, indicating the involvement of G_i- or G_o-type G proteins in the signaling pathways. Moreover, MCT-2 and hMCT-2 also stimulated β-hexosaminidase secretion in human neutrophils isolated from peripheral blood in a concentration-dependent manner. Additionally, these peptides partially competed with [³H]formyl-Met-Leu-Phe binding to HL-60 cells differentiated into neutrophilic/granulocytic cells, presenting the possibility that the receptor for MCT-2 and hMCT-2 is one of the formyl peptide receptors. These results demonstrate that MCT-2 and its human homolog hMCT-2 are cryptides that activate neutrophils, thus suggesting the presence of regulatory mechanisms involving such mitocryptides in innate immunity.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by research grants from the Special Research Project on Circulation Biosystem, University of Tsukuba; the Naito Foundation; the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 06680605); JT Inc.; and Mitsubishi Chemical.

² Address correspondence and reprint requests to Dr. Hidehito Mukai, 21st Century COE Program, Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina, Kyoto 607-8412, Japan. E-mail address: hmukai-endo{at}umin.ac.jp

³ T.S. and H.N. contributed equally to this study.

⁴ Current address: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan.

⁵ Abbreviations used in this paper: C5a, activated C component 5; MCT-1, mitocryptide-1; β-HA, β-hexosaminidase; AcOH, acetic acid; RP, reversed phase; ODS, octadecylsilane; TFA, trifluoroacetic acid; FAB, fast atom bombardment; MS, mass spectrometry; HBHS, HEPES-buffered Hanks’ solution containing 0.1% BSA; fMLF, formyl-Met-Leu-Phe; LDH, lactate dehydrogenase; [Ca²⁺]_i, the concentration of intracellular free Ca²⁺; PTX, pertussis toxin; pfCytB, porcine cytochrome b; MCT-2, mitocryptide-2; hMCT-2, the human homolog of MCT-2; FPR, formyl peptide receptor; FPRL1, FPR-like 1; FPRL2, FPR-like 2.