Purification and characterization of antimicrobial peptides from Skipjack tuna, Katsuwonus pelamis and Pacific oyster, Crassostrea gigas

Abstract

Part 1. A 2.3 kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative continuous acid-urea–polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin β-chain; thus, this peptide was designated as the Skipjack hemoglobin β chain-related antimicrobial peptide (SHβAP). SHβAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5-57.0 μg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus (MECs, 2.0-19.0 μg/mL), and against C. albicans (MEC; 12.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but protease and salt-sensitive. SHβAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α–helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin β–chain from marine fish. Part 2. An antimicrobial polypeptide was purified from an acidified gill extract of Pacific oyster (Crassostrea gigas) by C18 reversed-phase HPLC. The purified polypeptide had a molecular weight of 8,471 Da containing 74 amino acid residues. Comparison of the obtained N-terminal sequences with those of others revealed that it was identical to ubiquitin reported from other species and named cgUbiquitin. cgUbiquitin showed broad potent antimicrobial activity against Gram-positive and Gram-negative bacteria including Streptococcus iniae and Vibrio parahaemolyticus (minimal effective concentrations, 7.8 and 9.8 μg/mL), respectively, without hemolytic activity. The cgUbiquitin cDNA was identified from an EST library of oyster gill as a precursor form, encoding ubiquitin consisting of 76 amino acids fused to ribosomal protein of S27. Although the cgUbiquitin precursor mRNA was expressed at the intermediate level in the gill, the mRNA was significantly up-regulated at 48 h post injection with Vibrio sp. Analysis of the cgUbiquitin C-terminus by carboxypeptidase B treatment and comparison of the retention times revealed that cgUbiquitin lacks the terminal Gly-Gly doublet and ends in an C-terminal Arg residue which might be related to antimicrobial activity. Study of the kinetics of killing and membrane permeabilization showed that this peptide was not membrane permeable and acted through a bacteriostatic process. According to the homology modeling, this peptide is composed of three secondary structural motifs including three α-helices and four β-strands separated by 7 loops regions. Our results indicate that cgUbiquitin might be related to the innate immune defenses in the Pacific oyster and this is the first report for antimicrobial function of ubiquitin isolated from any oyster species.