Isolation, cDNA cloning and modification of defensin from the Coconut Rhinoceros Beetle, Oryctes rhinoceros

J. Ishibashi, H. Saido-Sakanaka, H. Tanaka, J. Yang & M. Yamakawa

National Institute of Sericultural and Entomological Science, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan

An antibacterial peptide, defensin, was purified by 4 steps of reversed phase HPLC from hemolymph of Oryctes rhinoceros larvae immunized with Escherichia coli. Staphylococcus aureus was used as an indicator bacteria. O. rhinoceros defensin showed antibacterial activity against Gram-positive but not against Gram-negative bacteria. The N-terminal amino acid sequence was determined with an amino acid sequencer. The O. rhinoceros defensin cDNA was cloned by 3 steps of polymerase chain reaction using fat body mRNA from immunized larvae. Deduced amino acid sequences from the nucleotide sequences indicated that the defensin contains a 79 amino acid precursor, in which the mature peptide (43 amino acids) is assumed to be produced by cleavage of the signal peptide and propeptide region by furin-like processing enzyme. Defensin from Allomyrina dichotoma and that from O. rhinoceros shared 86 % identity in the mature peptide region. The expression of O. rhinoceros defensin was induced in the fat body, hemocyte and Malpighian tubule after injection of E. coli. Expression was also observed in the midgut with and without the injection. This is assumed to be because the food compost always stimulates expression of antibacterial peptides. We synthesized a 9-mer fragment peptide based on O. rhinoceros defensin, which corresponds to the active site of A. dichotoma defensin. The fragment peptide and its analogues exhibited comparable activities to those of the A.dichotoma defensin based analogues. In vivo experiments were performed using methicillin resistant S. aureus (MRSA) infected mice. More than 50 % of the MRSA infected mice recovered injection of these peptides. These peptides did not stimulate super oxide synthesis from neutrophils nor NO synthesis from macrophages. This indicates the mechanism of recovery for these peptides is different from that of KLKLLLLLKLK-NH2, which binds to a cell surface receptor and activates the immune response of a neutrophil

Index terms: antibacterial peptide, Allomyrina dichotoma, fragment peptide, MRSA

More Topics on the Wing

About Us