Biomolecular identification of beta-defensin-like peptides from the skin of the soft-shelled turtle Apalone spinifera.

Numerous bacteria are frequently observed in the superficial corneocytes forming the corneous layer of the soft-shelled turtle Apalona spinifera. The resistance to bacterial penetration through the living epidermis in this turtle suggests the presence of an antimicrobial barrier, possibly derived from the presence of anti-microbial peptides in the epidermis. Four beta-defensin-like peptides, named As-BD-1 to 4, have been characterized from skin tissues using molecular and bioinformatics methods. The precursor peptides contain the beta-defensin motif with the typical cysteine localization pattern. The analysis of the expression for the four different beta-defensin-like proteins show that these molecules are expressed in the skin (epidermis and dermis) of the carapace, neck, digit, and tail but are apparently not expressed in the liver or intestine under normal conditions. These data suggest that in the skin of the soft-shelled turtle there are potential effective anti-microbial peptides against epidermal bacteria.

A novel beta-defensin structure: big defensin changes its N-terminal structure to associate with the target membrane.

Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. The amino acid sequence of big defensin is divided into an N-terminal hydrophobic domain and a C-terminal cationic domain, which are responsible for antimicrobial activities against Gram-positive and -negative bacteria, respectively. The N-terminal domain of big defensin forms a unique globular conformation with two alpha-helices and a parallel beta-sheet, while the C-terminal domain adopts a beta-defensin-like fold. Although our previous study implied that big defensin changes its N-terminal structure in a micellar environment, due to the poor quality of the NMR spectra it remained to be resolved whether the N-terminal domain adopts any structure in the presence of micelles. In this analysis, we successfully determined the structure of the N-terminal fragment of big defensin in a micellar solution, showing that the fragment peptide forms a single alpha-helix structure. Moreover, NMR experiments using paramagnetic probes revealed that the N-terminal domain of big defensin penetrates into the micelle with a dipping at the N-terminal edge of the alpha-helix. Here, we propose a model for how big defensin associates with the target membrane.

Defensins and the convergent evolution of platypus and reptile venom genes.

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermist's hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg. The platypus genome sequence provides a unique opportunity to unravel the evolutionary history of many of these interesting features. While searching the platypus genome for the sequences of antimicrobial defensin genes, we identified three Ornithorhynchus venom defensin-like peptide (OvDLP) genes, which produce the major components of platypus venom. We show that gene duplication and subsequent functional diversification of beta-defensins gave rise to these platypus OvDLPs. The OvDLP genes are located adjacent to the beta-defensins and share similar gene organization and peptide structures. Intriguingly, some species of snakes and lizards also produce venoms containing similar molecules called crotamines and crotamine-like peptides. This led us to trace the evolutionary origins of other components of platypus and reptile venom. Here we show that several venom components have evolved separately in the platypus and reptiles. Convergent evolution has repeatedly selected genes coding for proteins containing specific structural motifs as templates for venom molecules.

Identification of three novel ostricacins: an update on the phylogenetic perspective of beta-defensins.

Three new beta-defensins, ostricacins-2, 3 and 4 (Osp-2, 3 and 4), have been successfully purified and characterised from ostrich heterophils in addition to ostricacin-1 (Osp-1). These peptides are composed of 36-42 amino acids with a molecular weight range of 4.70-4.98 kDa. In vitro, Osp-1, 3 and 4 were active against Escherichia coli O157:H7 and Staphylococcus aureus 1056 MRSA, whilst Osp-2 was active against bacterial strains plus the yeast Candida albicans 3153A. Minimal inhibitory concentrations of the three ostricacins ranged from 0.96 microg/mL to 12.03 microg/mL. Comparison with the known beta-defensins from mammalian and other avian species revealed that the four ostricacins shared eight conserved residues (six cysteines and two glycines), identified as the 'beta-defensin core motif'. Comparisons of the sequence also indicated that beta-defensins could have originated from a common beta-defensin-like ancestor that occurred before avian and mammalian lines diverged.