br Materials and methods br Results br

Materials and methods
Results
Discussion In this study, we identified a novel AMP scolopendrasin X from Scolopendra subspinipes mutilans through genome analysis. Scolopendrasin X stimulated neutrophil activity, resulting in calcium increase, chemotactic migration, and superoxide anion production. Moreover, this novel AMP scolopendrasin X inhibited inflammatory cytokine production induced by LPS. We also demonstrated that scolopendrasin X-induced neutrophil activation was mediated by a chemoattractant receptor FPR2. Our results suggest that this novel AMP could be used as a material to stimulate innate immune response through neutrophil activation. Previously, we and others have demonstrated that FPR2, an important chemoattractant receptor, recognizes diverse extracellular ligands [24], including N-formyl peptides, endogenous acute phase reactant serum amyloid A, AMP LL37, eicosanoid lipoxin A4, and so on [21]. Among these various ligands, LL37 is an AMP that can directly kill bacterial pathogen [25]. LL37 is the only AMP that can bind to FPR2 up to date. In this study, we demonstrated that the novel AMP scolopendrasin X could stimulate FPR2. Scolopendrasin X stimulated intracellular calcium increase and chemotactic migration in FPR2-, but not in FPR1-or vector-expressing RBL-2H3 cells (Fig. 3B). Scolopendrasin X-induced and chemotactic migration were inhibited by WRW4, an FPR2 antagonist (Fig. 3C). These results suggest that scolopendrasin X is another AMP ligand that can bind to FPR2. Although scolopendrasin X and LL37 act on the same target receptor FPR2, the two AMPs share no conserved amino Preladenant sequences. Because physical characteristics of AMP are well-conserved in terms of amphipathic structure and positive charge, further study is needed to test whether these common characteristics of AMP are important for their recognition by the target receptor FPR2. Previously, we have reported that some AMPs isolated from Scolopendra subspinipes mutilans act on FPR1, but not FPR2 [26], [27]. However, the AMP identified in this study scolopendrasin X can bind to FPR2, but not FPR1 (Fig. 3A). Such differential activation of FPR1 and FPR2 by different AMPs isolated from Scolopendra subspinipes mutilans suggests that simple physical common characteristics such as amphipathic structure and a positive charge might not be enough to discriminate FPR1 and PFR2. Additional characteristics such as amino acid sequences would be important to be recognized differently by FPR1 or FPR2. Accumulating evidences have demonstrated that FPR2 is an important target for the development of therapeutic agents against diverse human disorders [24], [28]. Some FPR2 agonists have shown therapeutic effects against polymicrobial sepsis, ulcerative colitis, rheumatoid arthritis, and cancers [23], [24], [29]. In this study, we found that stimulation of FPR2 by a novel AMP scolopendrasin X elicited neutrophil activation, resulting in chemotactic migration and superoxide anion production (Fig. 1, Fig. 2). Since superoxide anion is an important weapon to kill invading pathogens, our results suggest that scolopendrasin X might have anti-microbial effect against bacterial infection. We also observed that scolopendrasin X significantly blocked inflammatory cytokine production induced by LPS in mouse neutrophils (Fig. 4). Because LPS-induced inflammatory cytokine production is an important factor that mediates pathogenesis of inflammatory diseases, our results suggest that scolopendrasin X might have anti-inflammatory activity against LPS. Collectively, our results suggest that this novel peptide might be used as an important material to control infectious or inflammatory diseases.
Acknowledgements This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: National Agricultural Genome Program, Project No. PJ01033804)” Rural Development Administration, Republic of Korea, and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2015R1A2A1A10054567).