cDNA cloning and functional characterisation of four antimicrobial peptides from Paa spinosa

2.1 Animals

Adult specimens of P. spinosa were captured in Lanxi, Zhejiang Province, China, and bred in captivity at Zhejiang Normal University. These frogs were used in this study. All experiments had been approved by Zhejiang Normal University.

2.2 Molecular cloning of cDNAs encoding the precursor proteins

Total RNA was extracted from the skin of the adult individuals using RNAiso Plus (Takara Bio, Dalian, China) according to the manufacturer’s instructions. cDNA synthesis was conducted using a high-fidelity PrimeScript RT-PCR kit (Takara Bio). The primers were designed according to the cDNA sequences of conserved N-terminal signal peptides from selected Ranidae AMP precursors and synthesised by Takara Bio Inc., First-strand cDNA was synthesised using the GSP1 primer (5′-ATGTTCACCTTGAAGAAATCCCTG-3′) and the first sense primer (5′-TACCGTCGTTCCACTAGATTT-3′). First-round PCR products were reamplified with the GSP1 primer and the second sense primer (5′-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3′). PCR was performed under the following conditions: 30 min at 94 °C, 30 s at 94 °C, 30 s at 56 °C and 1 min at 72 °C for 30 cycles; a final extension for 10 min at 72 °C was performed. PCR products were purified using 1% agarose gel, extracted using a SanPrep gel extraction kit (Sangon, Shanghai, China) and then cloned into the pMD-18T vector (Takara Bio) for sequencing.

2.3 Peptide synthesis

All peptides used in the bioactivity tests were synthesised using a peptide synthesiser (Model 433 A, Applied Biosystems Inc.) from GL Biochem Ltd. (Shanghai, China). The synthesised peptides were purified by reversed-phase HPLC with a Waters column (Waters ODS₂ C18, 0.46 cm×25 cm). The purity of the peptides was higher than 95% and their identity was confirmed by MALDI-TOF-MS (Voyager DE Pro, ABI). Prior to the assays, synthesised peptides were dissolved in sterile deionised water to a final concentration of 2 mg/mL.

2.4 Prediction of the physicochemical properties and amphipathicity of the AMPs

Peptide parameters, including net charge, theoretical pI, number of amino acids and grand average of hydropathicity (GRAVY), were computed by ProtParam. The GRAVY value for a protein is calculated as the sum of the hydropathy values of all of its amino acids [16]. Prediction of the secondary structures of the peptides was performed using the online HNN program (Hierarchical Neural Network Method of Guerm-eur, 1997) (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page¼/NPSA/npsa_hnn.html).

2.5 Antimicrobial assay

The bacterial strains used in the antimicrobial assays, including the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and the Gram-negative bacteria Escherichia coli and Aeromonas hydrophila, were obtained from the China General Microbiological Culture Collection Centre. Fresh LB medium was used in the culture of the microorganisms. The antibacterial activities of the peptides were tested using an inhibition zone assay on agarose plates seeded with viable bacteria according to Bennich et al. [17]. The minimum inhibitory concentration (MIC) of a peptide against the bacteria was determined by a standard micro-dilution method using 96-well microtitre plates [18]. Briefly, the peptide was serially diluted twofold to concentrations between 0.78 and 100 μg/mL in LB broth. About 50 μL of the samples were dispensed into the wells of the plate, and each solution was inoculated with 50 μL (2×10⁵ cfu/mL) of a bacterial culture in logarithmic growth phase. Sterile deionised water was used as a negative control. The absorbance of each sample at 600 nm was recorded, using a microtitre plate reader, after the cultures had been incubated for 18 h at 37 °C. The MIC was defined as the lowest peptide concentration present in a clear well determined by visual inspection. To assess the validity of the assay, parallel incubations were performed with gentamycin sulfate as a positive control.

2.6 Haemolytic assay

The haemolytic activity of the peptides was determined as follows. Approximately 6 mL of rabbit red blood cells (RBCs) was washed several times with 5 mL of 0.9% NaCl by centrifugation at 358 g for 5 min. Approximately 40 μL of the peptide samples (2 mg/mL) was added to 960 μL of diluted erythrocytes to obtain a final peptide concentration of 80 μg/mL, and the cell suspensions were incubated at 37 °C for 30 min. About μL of 0.1% Triton X-100 was added to 960 μL of diluted erythrocytes as a positive control, and 10 μL of 0.9% NaCl was added to 990 μL of diluted erythrocytes as a negative control. After 30 min, the solutions were centrifuged at 805 g for 5 min, and the absorbance in the supernatants was measured at 540 nm. The percentage of haemolysis, H%, was calculated according to the following equation: H% = (A_sample – A_negative) × 100/A_positive – A_negative. The experiments were carried out in triplicate.

2.7 Antioxidative activity

A free radical scavenging activity assay was used to examine the antioxidative activity of the peptides. 2,2-Diphenyl-1-picrylhydrazyl (DPPH; Sigma-Aldrich, St. Louis, MO, USA) was used as a stable radical, and the experiment was carried out as previously reported [19]. Briefly, 8 μL of peptide solution (0.2 mg/mL) was mixed with 92 μL of 6 × 10^–5 M DPPH dissolved in methanol. The mixture was kept in the dark for 30 min at room temperature, and the amount of reduced DPPH was quantified by measuring the decrease in absorbance at 520 nm. The percentage of free radicals scavenged (S%) was calculated according to the following equation: S% = (A_blank – A_sample) × 100/(A_blank). Deionised water was used as a negative control.

2.8 Cytotoxicity assay

The cytotoxicity of the peptides was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assay [20]. HeLa cells were donated by Zhejiang Normal University. About 200 μL of HeLa cells in the logarithmic growth phase were seeded into a 96-well plate and incubated at 37 °C for 24 h. The peptides were serially diluted to obtain final concentrations of 400, 200, 100, 50, 25 and 12.5 μg/mL. About 4 μL of each peptide concentration was dispensed into each well of the plate. Following incubation times of 20, 44 and 68 h, 20 μL of fresh medium containing 5 mg/mL MTT was added to the wells after removal of the test samples. After 4 h of incubation, 150 μL of DMSO was added to each well to dissolve the formed formazan crystals. The samples were subsequently shaken for 10 min at room temperature, and their absorbance was recorded at 570 nm using a microtitre plate reader. The positive control was a nutrient solution and the negative control was mitomycin. All experiments were performed three times with duplicates for each measurement point. The IC₅₀ value was determined as the concentration of a sample at which the absorbance at 570 nm was reduced by 50%.

3.1 Molecular cloning of cDNAs encoding AMP precursor proteins

A total of four cDNAs encoding the precursors of four AMPs were cloned from the P. spinosa skin cDNA library. Alignment of the amino acid sequences with the NCBI protein database revealed that the four AMPs represent a novel family of frog AMPs with no similarities to known amphibian AMPs, except for parkerin and japonicin-1NPa [21] (similarities of 61%–81%). According to the generic name of the species of origin, the peptides were named spinosan-A, spinosan-B, spinosan-C and spinosan-D. The nucleotide sequences of the cDNAs and deduced amino acid sequences are shown in Figure 1.

Figure 1:

Nucleotide and deduced amino acid sequences encoding AMP precursors. The box delineates the mature peptide and the asterisk indicates the stop codon.

All deduced precursor proteins showed overall architectures similar to those of other anuran AMP precursors. Each protein was composed of a putative signal peptide of 22 amino acid residues, an N-terminal acidic spacer domain of 16–29 residues, a Lys-Arg/Lys- processing site and a 12–23 residue C-terminal dermal peptide (Figure 2). The signal peptide sequences were highly similar amongst spinosan-A, spinosan-C and spinosan-D; in spinosan-B, Val¹¹ was replaced with Ile¹¹. The N-terminal acidic spacers of the peptides contained high levels of glutamic acid and aspartic acid.

Figure 2:

Amino acid sequence alignment of AMP precursor proteins of P. spinosa.

Region 1, putative signal peptide domain; region 2, variable length acidic “spacer” peptide domain; region 3, KR (-Lys–Arg-), the conserved classical pro-peptide convertase processing site; region 4, the hypervariable domain that encodes the mature AMP.

3.2 Analysis of the physicochemical properties and amphipathicity of the AMPs

The GRAVY, net charge, theoretical pI and number of amino acids of the AMPs are shown in Table 1. Amongst the AMPs obtained in this study, only spinosan-C showed cationic features with a hydrophobic property.

3.3 Antimicrobial assay

The antimicrobial activities of the synthetic peptides were determined as MIC values. At a dose of 100 μg/mL, only spinosan-D exhibited slight anti-microbial activities towards Gram-positive and Gram-negative bacteria. Even at 2 mg/mL, spinosan-A, spinosan-B and spinosan-C showed no antimicrobial activity.

3.4 Haemolytic assay

The potential toxicity of AMPs was measured by a haemolytic assay against rabbit RBCs. No haemolysis was observed in the negative control, while 100% haemolysis was observed in the positive control (Triton-X). At a concentration of 80 μg/mL of spinosans A-D, maximally 4% haemolyis of rabbit RBC was observed (Table 2). This finding indicates that the interactions between the four AMPs and the phospholipids of the cell membrane of RBCs are very weak.

3.5 Antioxidative activity

At a concentration of 80 μg/mL, spinosan-C exhibited apparent antioxidative activity with an S% value-of 85%. For spinosans A, B and D the respective values were 52, 59 and 49%, respectively (Figure 3).

Figure 3:

DPPH radical scavenging activity of spinosans A–D at a concentration of 80 μg/mL.

3.6 Cytotoxicity assay

The four AMPs exhibited low cytotoxic activity against HeLa cells. The cytotoxicity of spinosan-A was higher than that of the three other AMPs (Table 3).