Abhisin: A potential antimicrobial peptide derived from histone H2A of disk abalone (Haliotis discus discus) Mahanama De Zoysa a , Chamilani Nikapitiya a , Ilson Whang b , Jae-Seong Lee c , Jehee Lee a, d, * a Department of Marine Life Science, College of Ocean Science, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea b Department of Life Sciences, College of Natural Science, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea c Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea d Marine and Environmental Institute, Jeju National University, Jeju Special Self-Governing Province 690-814, Republic of Korea article info Article history: Received 1 June 2009 Received in revised form 9 August 2009 Accepted 11 August 2009 Available online 23 August 2009 Keywords: Abalone Abhisin Antimicrobial peptide Histone H2A Invertebrate abstract Antimicrobial peptides (AMPs) play an important role in the immune defense against pathogenic microor- ganisms. In this study, a histone H2A full-length cDNA was cloned from disk abalone Haliotis discus discus. We identified a 40-amino acid AMP designated as abhisin from the N-terminus of the abalone histone H2A sequence. Abhisin shows the characteristic features of AMPs including net positive charge (þ13), higher hydrophobic residues (27%) and 2.82 kcal/mol protein binding potential. Abhisin shares 80% amino acid identity with the buforin I peptide that is derived from Asian toad histone H2A. We synthesized the synthetic peptide of abhisin, and characterized its antimicrobial activities. Our results showed the growth inhibition of Gram positive (Gþ) Listeria monocytogenes, Gram negative (G-) Vibrio ichthyoenteri bacteria, and fungi (yeast) Pityrosporum ovale by abhisin treatment at 250 mg/mL. However, stronger activity was displayed against the Gþ than G bacteria. Additionally, scanning electron microscope (SEM) observation results confirmed that P. ovale cells were damaged by abhisin treatment. Interestingly, abhisin treatment (50 mg/mL) decreased the viability of THP-1 leukemia cancer cells approximately by 25% but there was no effect on the normal vero cells, suggesting that abhisin has cytotoxicity against cancer cells but not normal cells. Quantitative real time RT-PCR results revealed that histone H2A transcription was significantly induced at 3 h post-infection with bacteria in abalone gills and digestive tract. These results suggest that abhisin is a potential antimicrobial agent, and its precursor histone H2A may be involved in the innate immune defense system in abalone. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Antimicrobial peptides (AMPs) are major components of host defense systems of plants, invertebrates and vertebrates against invading pathogens [1,2]. Most AMPs are membrane-active hydro- phobic and positively charged (cationic) molecules of less than 10 kDa in molecular mass. The net positive charges of AMPs promote their binding to the membranes of microbes, which generally have an overall negative charge [3,4]. Recent studies have demonstrated the importance of several AMPs as their high biochemical diversity, broad specificity against microorganisms [5], anticancer activities [6], wound healing effects [7], regulation of cell proliferation, extra cellular matrix production, anti-endotoxin activities and cellular immune responses by inducing gene expression [8,9]. Several reports have shown that histone protein or histone- derived peptides process antimicrobial activity in vertebrates including human [10,11], amphibian [12,13] and fish [14,15]. The first histone antimicrobial activity was demonstrated from calf thymus in 1958 [10]. Buforin I was the first histone H2A-derived AMP from the Asian toad [12]. Other histone-derived AMPs have been identi- fied from fish including catfish Parasilurus asotus [16], Atlantic salmon Salmon salar [17], rainbow trout Oncorhynchus mykiss [14], and Atlantic halibut Hippoglossus hippoglossus [15]. However, only few histone-derived AMPs have been identified from invertebrates such as pacific white shrimp [18] and scallop Chlamys farreri [19]. Until the present time, AMPs have never been characterized in detail in abalone, despite two sequences of defensin from Haliotis discus discus (FJ864724) and H. discus hannai (ABF69125) available at GenBank. In this study, we report on the histone H2A-derived AMP named as ‘‘abhisin’’ from disk abalone H. discus discus. To understand the antimicrobial potential of abhisin, we synthesized a synthetic abhi- sin peptide, and determined its spectrum of activity against bacteria and fungi (yeast). Additionally, the morphological changes of yeast * Corresponding author at: Department of Marine Life Science, College of Ocean Science, Jeju National University, Jeju 690-756, Republic of Korea. Tel.: þ82 64 754 3472; fax: þ82 64 756 3493. E-mail address: jehee@jejunu.ac.kr (J. Lee). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.08.007 Fish & Shellfish Immunology 27 (2009) 639–646