Bombinin-like peptide 7

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Bombinins are a group of amphibian-derived peptides with broad spectrum antimicrobial activities that have been only identified from the ancient toad species, Bombina. In this study, we described the identification and characterization of a novel bombinin precursor which encoded a bombinin-like peptide (BLP-7) and a novel bombinin H-type peptide (named as Bombinin H-BO) from the skin secretion of Oriental fire-bellied toad, Bombina orientalis. The primary structures of both mature peptides were determined by combinations of molecular cloning of peptide precursor-encoding cDNAs and mass spectrometry techniques. Secondary structure prediction revealed that both peptides had cationic amphipathic α-helical structural features. The synthetic replicate of BLP-7 displayed more potent antimicrobial activity than Bombinin H-BO against Gram-positive and Gram-negative bacteria and yeast. Also, in vitro antitumour assay showed that both peptides possessed obvious antiproliferative activity on three human hepatoma cells (Hep G2/SK-HEP-1/Huh7) at the non-toxic doses. These results indicate the peptide family of bombinins could be a potential source of drug candidates for anti-infection and anticancer therapy.

The structure of a gene coding for bombinin-like peptides (BLP) in Bombina orientalis was determined. It comprises two exons separated by a 1337 bp intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for BLP-7 and a bombinin H-type peptide (GH-2). The promoter region contains putative recognition sites for nuclear factors, such as NF-IL6 and NF-kappaB. The analysis of the structure of this gene, compared with that of the previously reported BLP-3 gene sequence, suggests the occurrence of a gene duplication event, rather than an alternative splicing mechanism, which leads to the generation of both inter- and intra-families variability in this class of cytolytic peptides. Furthermore, chromosome walking analysis indicates that this gene family is not densely clustered.

Propionibacterium acnes (P. acnes) is a Gram-positive commensal bacterium, which is involved in the pathogenesis and inflammation of acne vulgaris. An antimicrobial peptide named bombinin-like peptide 7 (BLP-7), which was determined from Bombina orientalis, has been shown to possess certain antibacterial activity. This study was carried out with synthesized BLP-7 on the basis of the antimicrobial and anti-inflammatory activities against P. acnes in vitro and in vivo. The minimal inhibitory concentration (MIC) of BLP-7 against P. acnes is 5 μM. And BLP-7 exhibits strong resistance to heat, pH and salt concentration, but no significant cytotoxicity to normal human epidermal keratinocytes (NHEKs). Using the co-culture of P. acnes and NHEKs, this study demonstrated that BLP-7 significantly reduced the production of interleukin (IL)-8 and granulocyte-macrophage colony stimulating factor (GM-CSF), as well as the expression of these two pro-inflammatory cytokines at the transcriptional level. In a separate study, using the rat ear edema model, BLP-7 significantly suppressed P. acnes-induced skin inflammation, reducing the ear thickness by 54.21% of the negative control group. These results suggest that due to its anti-P. acnes and anti-inflammatory activities, BLP-7 could be used as a potential anti-acne agent.