Open AccessPurification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros
Author(s) -
Ishibashi Jun,
SaidoSakanaka Hisako,
Yang Jun,
Sagisaka Aki,
Yamakawa Minoru
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00906.x
Subject(s) - defensin , complementary dna , antibacterial activity , biology , midgut , escherichia coli , microbiology and biotechnology , peptide sequence , peptide , staphylococcus aureus , signal peptide , biochemistry , bacteria , gene , botany , larva , genetics
A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros , immunized with Escherichia coli . A full‐size cDNA was cloned by combining reverse‐transcription PCR (RT‐PCR), and 5′‐ and 3′‐rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus . A 9‐mer peptide amidated at its C‐terminus, AHCLAICRK‐NH 2 (Ala22–Lys30‐NH 2 ), was synthesized based on the deduced amino‐acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma . This peptide showed antibacterial activity against S. aureus , methicillin‐resistant S. aureus , E. coli and Pseudomonas aeruginosa . We further modified this oligopeptide and synthesized five 9‐mer peptides, ALRLAIRKR‐NH 2 , ALLLAIRKR‐NH 2 , AWLLAIRKR‐NH 2 , ALYLAIRKR‐NH 2 and ALWLAIRKR‐NH 2 . These oligopeptides showed strong antibacterial activity against Gram‐negative and Gram‐positive bacteria. The antibacterial effect of Ala22–Lys30‐NH 2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome‐entrapped glucose. These Ala22–Lys30‐NH 2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR‐NH 2 .