Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity Akihiro Wada a,⇑ , Pooi-Fong Wong b , Hironobu Hojo c , Makoto Hasegawa d , Akitoyo Ichinose e , Rafael Llanes f , Yoshinao Kubo g , Masachika Senba h , Yoshio Ichinose i a Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan b Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, Kanagawa 2591292, Japan d Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Shiga 5260829, Japan e Electron Microscopy Shop Central Laboratory, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan f Institute Pedro Kouri, Havana, Cuba g Division of Cytokine Signaling, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 8528523, Japan h Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan i Kenya Research Station, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan article info Article history: Received 11 March 2013 Available online 26 March 2013 Keywords: Alarin Galanin LL-37 Antimicrobial peptide Escherichia coli Staphylococcus aureus Hemolysis Membrane blebbing abstract Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicro- bial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus . Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction In recent years, research in the field of novel antimicrobial pep- tides has intensified due to the need for newer effective antibiotics to overcome resistance issues of conventional antibiotics. Antimi- crobial peptides, isolated from various bacteria, fungi, plants, invertebrates and vertebrates are important components of natural defenses of most living organisms [1]. These molecules have the added advantages of being very small in size, amphipathic and pos- itively charged which allow them to bind and disrupt microbial membranes [2]. Some effective antimicrobial peptides reported thus far include human LL37 which is active against Staphylococcus aureus [3,4] and Escherichia coli [5] and b-defensins against Entero- coccus faecalis and Helicobacter pylori [6–9]. The galarin family of neuropeptides consists of galanin, a gala- nin-like peptide (GALP) and a newer member called alarin. Human galanin consists of 30 amino acids and is encoded by the GAL gene. GALP consists of 60 amino acids and it can activate galanin recep- tors (GalRs) because residues 9–21 of GALP are identical to the first 13 amino acids of galanin [10]. Alarin consists of 25 amino acids and is derived from an alternative-splicing of the GALP gene that excludes exon 3. Its precursor consists of the signal sequence of the prepro-GALP, the first 5 amino acids of the mature GALP pep- tide and another 20 amino acids that are not identical to any other peptides. Unlike galanin and GALP, alarin does not bind to GalRs [11,12]. Galanin and GALP mRNAs are widely distributed in CNS as well as in the periphery in GIT, heart, dermis, epidermis, nerves, bone and joint tissues [13]. Alarin mRNA was first detected in ganglionic cells of neuroblastonic tumors [11] and it has a much wider CNS distribution than GALP [14]. It can also be found localized around blood vessels in the skin [11]. Galanin and GALP are involved in a diverse range of normal brain functions such as feeding and 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.03.045 Abbreviations: GALP, galanin-like peptide; E. coli , Escherichia coli ; S. aureus , Staphylococcus aureus . ⇑ Corresponding author. Fax: +81 95 819 7805. E-mail address: a-wada@nagasaki-u.ac.jp (A. Wada). Biochemical and Biophysical Research Communications 434 (2013) 223–227 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc