EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 44, No. 2, 130-137, February 2012 Identification of novel peptides that stimulate human neutrophils Geon Ho Bae1*, Ha Young Lee1,2*, Young Su Jung1,2, Jae Woong Shim1, Sang Doo Kim1,2, Suk-Hwan Baek3, Jae Young Kwon1, Joon Seong Park4 and Yoe-Sik Bae1,2,5 1Department of Biological Sciences Sungkyunkwan University Suwon 440-746, Korea 2Mitochondria Hub Regulation Center College of Medicine, Dong-A University Busan 602-714, Korea 3Department of Biochemistry and Molecular Biology Yeungnam University Daegu 705-030, Korea 4Department of Hematology-Oncology Ajou University School of Medicine Suwon 443-721, Korea 5Corresponding author: Tel, 82-31-290-5914; Fax, 82-31-290-7015; E-mail, yoesik@skku.edu *These authors contributed equally to this work. http://dx.doi.org/10.3858/emm.2012.44.2.008 Accepted 14 November 2011 Available Online 16 November 2011 Abbreviations: FPR, formyl peptide receptor; GMMWAI, Gly- Met-Met-Trp-Ala-Ile-CONH2; IP3, inositol-1,4,5-triphosphate; MMHWAM, Met-Met-His-Trp-Ala-Met-CONH2; MMHWFM, Met-Met-His-Trp-Phe-Met-CONH2; PLC, phospholipase C; PS-SPCL, positional scanning synthetic peptide combinatorial library; PTX, pertussis toxin Abstract Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we iden- tified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified pep- tides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-de- pendent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leu- kocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimu- lated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide re- ceptor (FPR)1 while the other peptide (MMHWAM) act- ed on FPR2. Since the three novel peptides were specif- ic agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling. Keywords: calcium; chemotaxis; neutrophils; pep- tide library; peptides; receptors, formyl peptide Introduction Neutrophils play an important role in the regulation of immune responses, especially in the innate immune response (Kumar and Sharma, 2010). Neutrophils are recruited to infected areas or sites of injury according to a chemoattractant gradient (Borregaard, 2010; Kumar and Sharma, 2010). Recruited neutrophils can be activated by extra- cellular stimuli, resulting in the production of several reactive oxygen species (Dahlgren and Karlssson, 1999; Walther et al., 2000; Tiffany et al., 2001). It is a critical aspect of resting neutrophils that they must be activated for host defense. The activation of neutrophils can be induced by various different stimuli, including pathogen-derived molecules and several chemoattractants (Walther et al., 2000; Tiffany et al., 2001; Sabroe et al., 2003; Kobayashi, 2008). Among these, chemo- attractants, including several chemokines that regulate the activities of neutrophils, have received much attention over the last decade. These chemoattractants bind to specific cell surface receptors, which are coupled to pertussis toxin (PTX)-sensitive G-protein(s), resulting in intracel- lular Ca2+ mobilization, cell migration, exocytosis, adhesion, and generation of bioactive lipids and/or reactive oxygen species (Walther et al., 2000; Tiffany et al., 2001; Kobayashi, 2008). Keeping in mind the important roles of chemoattractants in Copyright ⓒ 2012 by the The Korean Society for Biochemistry and Molecular Biology This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.