Isolation and Characterization of a Defensin-Like Peptide (Coprisin) from the Dung Beetle, Copris tripartitus

Abstract

The antibacterial activity of immune-related peptides, identified by a differential gene expression analysis, was investigated to suggest novel antibacterial peptides. A cDNA encoding a defensin-like peptide, Coprisin, was isolated from bacteria-immunized dung beetle, Copris tripartitus, by using differential dot blot hybridization. Northern blot analysis showed that Coprisin mRNA was up-regulated from 4 hours after bacteria injection and its expression level was reached a peak at 16 hours. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with a predicted molecular weight of 8.6 kDa and a pI of 8.7. The amino acid sequence of mature Coprisin was found to be 79.1% and 67.4% identical to those of defensin-like peptides of Anomala cuprea and Allomyrina dichotoma, respectively. We also investigated active sequences of Coprisin by using amino acid modification. The result showed that the 9-mer peptide, LLCIALRKK-NH(2), exhibited potent antibacterial activities against Escherichia coli and Staphylococcus aureus.

Figure 1

Isolation of Coprisin cDNA. (a) Differential dot blot hybridization. Randomly selected 1862 cDNA clones from cDNA library of Copris tripartitus larvae were hybridized with probes of E. coli-immunized larvae. In this figure, one pair of hybridization dot blots is shown for the Coprisin cDNA clone (adapted from Hwang et al. [13]). The arrow indicates a differentially expressed Coprisin cDNA clone in saline- or E. coli-immunized larvae. (b) Northern blot hybridization of the Coprisin gene for a time-course after immunization. C. tripartitus larvae were injected with 50 μL of E. coli JM109 (5 × 10⁵ cells) suspended in physiological saline (150 mM NaCl/5 mM KCl). Larvae were kept for 0, 4, 8, 16, and 24 hours. Ten microgram aliquots of total RNA were resolved on formaldehyde containing agarose gels and blotted onto nitrocellulose membranes. The probe was labeled with [α-³²P]dCTP. As an internal marker, 28S rRNA was stained with ethidium bromide.

Figure 2

Full-length cDNA sequence and comparison of amino acid sequences of insect defensins. (a) Nucleotide and deduced amino acid sequences of Coprisin gene isolated from C. tripartitus. The predicted amino acid sequence (single-letter abbreviation) is shown below the nucleotide within the open reading frame (ORF). The potential recognition sequence for the cleavage site within the constitutive secretory pathway (Arg-Xaa-Lys/Arg-Arg) is single-underlined. The putative mature peptide is boxed. Asterisk indicates the termination codon. Codons for initiation, termination, polyadenylation, and poly(A) tail are in bold. (b) Comparison of seven insect defensins sequences. Bars indicate gaps to optimize the sequence alignment. The six observed Cys(C) residues, which make up the disulphide bridges, are shaded grey. The GenBank accession numbers for the analyzed sequence are Anomala cuprea (BAD77966), Oryctes rhinoceros (BAA36401), Allomyrina dichotoma (AAB36306), Copris tripartitus (ABP97087), Thermobia domestica (CAM36306), Acalolepta luxuriosa (AAK35160), Tenebrio molitor (BAA04552), and Tribolium castaneum (XP968237).