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. 2020 May 29;40(5):BSR20200019.
doi: 10.1042/BSR20200019.

Brevinin-1GHd: a novel Hylarana guentheri skin secretion-derived Brevinin-1 type peptide with antimicrobial and anticancer therapeutic potential

Yangyang Jiang  1 , Yue Wu  1 , Tao Wang  1 , Xiaoling Chen  1 , Mei Zhou  1 , Chengbang Ma  1 , Xinping Xi  1 , Ying Zhang  1   2 , Tianbao Chen  1 , Chris Shaw  1 , Lei Wang  1
Affiliations

Brevinin-1GHd: a novel Hylarana guentheri skin secretion-derived Brevinin-1 type peptide with antimicrobial and anticancer therapeutic potential

Yangyang Jiang et al. Biosci Rep. .

Abstract

Host-defense antimicrobial peptides (AMPs) from amphibians are usually considered as one of the most promising next-generation antibiotics because of their excellent antimicrobial properties and low cytotoxicity. In the present study, one novel Brevinin-1 type peptide, Brevinin-1GHd, was isolated and characterized from the skin secretion of the frog, Hylarana guentheri. Brevinin-1GHd was found to possess a wide range of antimicrobial activity through penetrating the bacterial membrane within a short time while showing low hemolysis at bactericidal concentrations, even against the resistant strains. It also inhibited and eradicated biofilms that are thought to be closely related to the rise in resistance. Meanwhile, Brevinin-1GHd exhibited wide-spectrum anti-proliferation activity toward human cancer lines. Taken together, these results indicate that Brevinin-1GHd with its excellent antimicrobial and anticancer activities is a promising candidate for a novel antibiotic agent, and study of its structure-activity relationships also provided a rational template for further research and peptide analog design.

Keywords: Brevinin-1GHd; anti-proliferation; antimicrobial peptides (AMPs); biofilms; resistant strains.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Nucleotide and translated open reading frame amino acid sequence of cDNA encoding the Brevinin-1GHd peptide precursor cloned from the skin secretion library of Hylarana guentheri
The putative signal peptide (double-underlined), mature peptide (single-underlined) and stop codon (asterisk), are indicated.
Figure 2
Figure 2. Chromatogram of Hylarana guentheri skin secretion and corresponding mass spectra of Brevinin-1GHd
(A) Region of RP-HPLC chromatogram of Hylarana guentheri skin secretion with the elution position/retention time of Brevinin-1GHd indicated by an arrow. Absorbance wavelength was set at 214 nm. (B) MALDI-TOF mass spectrum of the HPLC fraction at 50 min in panel (A) corresponding to Brevinin-1GHd. (C) Thermoquest LCQ™ fragment scan spectrum derived from ions corresponding to Brevinin-1GHd. Observed ions following MS/MS fragmentation are indicated in color.
Figure 3
Figure 3. Bioinformatics and secondary structure analyses of Brevinin-1GHd
(A) Alignment of mature peptide Brevinin-1GHd with similar AMPs of the Brevinin-1 subfamily found in the NCBI database. Identical amino acids residues are indicated by asterisks. (B) Helical wheel projection of Brevinin-1GHd with an arrow indicated the hydrophobic face. (C) Predicted secondary structure with a dominated alpha-helix structure. (D) Secondary structures of Brevinin-1GHd. The CD spectra of peptides were measured in an aqueous environment (10 mM NH4AC buffer) and a membrane-mimetic environment (50% TFE in 10 mM NH4AC), respectively.
Figure 4
Figure 4. Time-kill curves of Brevinin-1GHd
Responses of 2-fold MICs (green), 1-fold MICs and 0.5-fold MICs (grey) of peptide over 120 min were plotted against four different strains (A) E. coli, (B) S. aureus, (C) P. aeruginosa and (D) MRSA. The X-axis is time in min, the Y-axis is CFU/ml. AMP (blue) and control (red) represent Ampicillin (32 µM) and control group (no drug), respectively.
Figure 5
Figure 5. Cell permeability of S. aureus, MRSA and E. coli treated with Brevinin-1GHd at 1-fold, 2-fold and 4-fold of MIC
The cells permeabilized by 70% isopropanol were used as the positive control (P). *P < 0.05 versus MRSA incubated with 1-fold MIC of Brevinin-1GHd.
Figure 6
Figure 6. Hemolytic and cytotoxic activity of Brevinin-1GHd
(A) Hemolytic activity of Brevinin-1GHd. The highest MIC/MBC value of the tested bacteria P. aeruginosa (32 µM) is indicated by the red arrow. P and B represent positive control (1% Triton X-100) and negative control (PBS), respectively. (B) Dose-dependent anti-proliferative effects of Brevinin-1GHd against HMEC-1 cells (left) and HaCaT cells (right); *P < 0.05, ***P < 0.001, ****P < 0.0001 in comparison with control group (no drug).
Figure 7
Figure 7. Anti-proliferation activity of Brevinin-1GHd on human cancer cell lines
(A) H157, (B) U251MG, (C) MDA-MB-435s and (D) PC3. Each cell line was treated with Brevinin-1GHd in a range of concentrations from 10−10 to 10−4 M. N means negative control group (no drug). ****P < 0.0001 in comparison with negative control group.
Figure 8
Figure 8. Comparison of hemolytic activity between Brevinin-1GHa and Brevinin-1GHd
Figure 9
Figure 9. Sequence diversity in the “Rana-Box” loop of heptapeptide-ring containing Brevinin-1 family peptides
This heptapeptide loop sequences were obtained from previous reported Brevinin-1 family peptides (those containing heptapeptide rings) alignment illustrated with a sequence logo graphic. The X-axis demonstrates the diversity of residues at each position and the Y-axis shows the relative frequency.
See this image and copyright information in PMC

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