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
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
antibacterial peptide, Allomyrina dichotoma, fragment