Peptide name : Bombinins BLP-7/GH-2

Source/Organism : Oriental fire-bellied toad

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 144

C-terminal modification: Not found

N-terminal modification : Amidation

Non-natural peptide information: None

Assay type : In vitro antitumor assay

Assay time : 24h

Activity : IC50 : 2.88 μM

Cell line : HepG2

Cancer type : Human hepatoma

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 16053.2366 Dalton

Aliphatic index : 0.962

Instability index : 44.0007

Hydrophobicity (GRAVY) : -0.348

Isoelectric point : 6.7644

Charge (pH 7) : -0.2859

Aromaticity : 0.055

Molar extinction coefficient (cysteine, cystine): (4470, 4470)

Hydrophobic/hydrophilic ratio : 0.94594594

hydrophobic moment : -0.436

Missing amino acid : C,W

Most occurring amino acid : L

Most occurring amino acid frequency : 19

Least occurring amino acid : H

Least occurring amino acid frequency : 2

Secondary structure fraction (Helix, Turn, Sheet): (0.4, 0.2, 0.3)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)CNC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCSC)[C@@H](C)CC)C(C)C)C(C)C)[C@@H](C)CC)[C@@H](C)O)C(C)C)C(C)C)[C@@H](C)CC)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)NCC(=O)O)[C@@H](C)CC)C(C)C)C(C)C)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)O)C(C)C)C(C)C)[C@@H](C)O

1 : Miele R, et al. Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7. Peptides. 2000; 21:1681-6. doi: 10.1016/s0196-9781(00)00317-x

2 : Ke T, et al. A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes. BMC Biotechnol. 2012; 12:10. doi: 10.1186/1472-6750-12-10

3 : Wu Y, et al. Inhibitory effect of the antimicrobial peptide BLP-7 against Propionibacterium acnes and its anti-inflammatory effect on acne vulgaris. Toxicon. 2020; 184:109-115. doi: 10.1016/j.toxicon.2020.06.006

4 : Zhou C, et al. Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire-bellied toad, Bombina orientalis. Chem Biol Drug Des. 2018; 91:50-61. doi: 10.1111/cbdd.13055

Paper title : Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7.

Doi : https://doi.org/10.1016/s0196-9781(00)00317-x

Abstract : 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.

Paper title : A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes.

Doi : https://doi.org/10.1186/1472-6750-12-10

Abstract : BACKGROUND: To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity. RESULTS: Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia. CONCLUSIONS: The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.

Paper title : Inhibitory effect of the antimicrobial peptide BLP-7 against Propionibacterium acnes and its anti-inflammatory effect on acne vulgaris.

Doi : https://doi.org/10.1016/j.toxicon.2020.06.006

Abstract : 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.

Paper title : Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire-bellied toad, Bombina orientalis.

Doi : https://doi.org/10.1111/cbdd.13055

Abstract : 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.