Cloning and Functional Analysis of Skin Host Defense Peptides from Yakushima Tago's Brown Frog (Rana tagoi yakushimensis) and Development of Serum Endotoxin Detection System

Abstract

Background/Objective: Amphibian skin is a valuable source of host defense peptides (HDPs). This study aimed to identify HDPs with novel amino acid sequences from the skin of Rana tagoi yakushimensis and analyze their functions. Methods: cDNAs encoding HDP precursors were cloned and sequenced using RT-PCR and 3'-RACE. The novel HDPs were synthesized to evaluate their antimicrobial activity, antioxidant activity, and cytotoxicity. Antimicrobial activity was evaluated by way of broth microdilution and endotoxin- and β-glucan-binding capacity using an enzyme-linked endotoxin binding assay (ELEBA) and a modified ELEBA, respectively. Results: Nine cDNAs encoding precursors for various HDP families, including temporin, ranatuerin-2, brevinin-1, amurin-9, and a novel yakushimin peptide, were identified. Brevinin-1TYa exhibited antibacterial activity against Staphylococcus aureus, and brevinin-1TYa and amurin-9TYa induced morphological changes in Escherichia coli and S. aureus. Yakushimin-TYa, amurin-9TYa, and brevinin-1TYa showed concentration-dependent antibacterial effects against the plant pathogens Xanthomonas oryzae pv. oryzae and Clavibacter michiganensis subsp. michiganensis. Amurin-9TYa demonstrated strong binding affinity to lipopolysaccharide, lipoteichoic acid, and β-glucan, exhibited antioxidant activity, and lacked cytotoxicity, making it a promising therapeutic candidate. Moreover, brevinin-1TYa showed strong cytotoxicity, whereas yakushimin-TYa exhibited weak cytotoxicity. Conclusions: These findings highlight the potential of these peptides, particularly amurin-9TYa, for future applications as antimicrobial and therapeutic agents.

Keywords: ELEBA; ELGBA; Rana tagoi yakushimensis; amurin-9; anti-plant pathogenic activity; antioxidant activity; brevinin-1; frog skin; host defense peptides; yakushimin.

Figure 1

Nucleotide and deduced amino acid sequences of HDP precursor cDNAs that were cloned from R. t. yakushimensis skin; in the nucleotide sequences, the forward and reverse primer-derived sequences are underlined. The putative polyadenylation signals are indicated with gray In the amino acid sequences, the forward primer-derived and HDP sequences are indicated with blue and red letters, respectively. Stop codons are marked with asterisks (*), and cleavage sites for signal peptidase and processing enzymes are marked with slashes (/) and boxes, respectively.

Figure 2

Comparison and schematic alignment of the amino acid sequences of the deduced mature temporin, ranatuerin-2, brevinin-1, amurin-9, and yakushimin peptides, as well as other peptides that exhibited the highest sequence similarities in a BLAST search. Residues that are conserved among each peptide are marked by the same color. The reference sequences of each peptide family are provided at the top of each block; generally, these are the originally isolated sequences. Gaps (-) were introduced to maximize sequence identities. Species names (except for R. t. yakushimensis peptides) and the values of the deduced net positive charge and pI are shown in parentheses.

Figure 3

(A) Effects of various concentrations of yakushimin-TYa (blue), amurin-9TYa (green), and brevinin-1TYa (orange) on the growth of Escherichia coli and Staphylococcus aureus. Bacterial cells were incubated with twofold serially diluted peptides for 20 h at 37 °C. Points and vertical bars represent the mean ± standard error of the mean (SE); n = 4. Values with the same superscripts are not significantly different (p ≥ 0.05). (B) Scanning electron microscopy (SEM) analysis of E. coli and S. aureus cells treated with yakushimin-TYa, amurin-9TYa, or brevinin-1TYa. Bacterial cultures in the mid-log phase were incubated with the various peptides (128 μg/mL) for 1 h at 25 °C and examined by SEM. Abnormally shaped cells indicating cell destruction (arrows) are visible in amurin-9TYa-treated E. coli and S. aureus, and brevinin-1TYa-treated S. aureus, compared to the myoglobin-treated controls. Scale bar = 1 μm.

Figure 4

Effects of various concentrations of yakushimin-TYa (blue), amurin-9TYa (green), and brevinin-1TYa (orange) on the growth of plant pathogenic Gram-negative bacteria Xanthomonas oryzae pv. oryzae and Gram-positive bacteria Clavibacter michiganensis subsp. michiganensis. Cells of each bacterial strain were incubated with serially diluted yakushimin-TYa, amurin-9TYa, or brevinin-1TYa peptides for 24 h at 28 °C. Points and vertical bars represent the mean ± SE (n = 4). In the panels, values with the same superscript are not significantly different (p ≥ 0.05).

Figure 5

(A) Evaluation of the binding abilities of yakushimin-TYa (blue), amurin-9TYa (green), and brevinin-1TYa (orange) to the bacterial endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA) using an enzyme-linked endotoxin binding assay (ELEBA). Twofold serially diluted peptides were coated onto 96-well Immobilizer plates and incubated with biotin-LPS or biotin-LTA for 4 h at 25 °C. Detection was performed using streptavidin-conjugated horseradish peroxidase (SA-HRP) and TMB substrate, and absorbance was measured at 450 nm. (B) Assessment of the competitive binding of LPS (dark green) and LTA (light green) to amurin-9TYa and verification in serum. Amurin-9TYa (40 µg/mL) was incubated on Immobilizer plates at 4 °C for 16 h. Biotin-LPS was mixed with LPS in PBS or FBS (500 ng/mL), and biotin-LTA was mixed with non-labeled LTA, both in blocking reagents. For the competition assays with LTA in FBS, a chloroform extraction was performed. The mixture was stirred and centrifuged, and the supernatant was collected, vaporized, and redissolved in endotoxin-free water for the competition experiment. Columns and vertical bars represent the mean ± SE (n = 4). Bovine serum albumin (BSA; white) was used as a control. Values with the same letters are not significantly different (p ≥ 0.05).

Figure 6

Evaluation of the binding abilities of yakushimin-TYa (blue), amurin-9TYa (green), and brevinin-1TYa (orange) to the fungal cell surface substance β-glucan using a modified ELEBA method, the enzyme-linked β-glucan-binding assay (ELGBA). This method replaces the biotin-labeled endotoxins in the ELEBA method with biotin-labeled β-glucan. The other difference is that the reaction with SA-HRP was extended to 40 min and that with the TMB chromogenic solution to 25 min. Other reaction conditions and details of the preparation of biotin-labeled β-glucan are described in the Section 4. Columns and vertical bars represent the mean ± SE (n = 4). BSA (white) was used as a control. In each panel, values with the same letters are not significantly different (p ≥ 0.05).

Figure 7

Evaluation of antioxidant activities of yakushimin-TYa (blue), amurin-9TYa (green), and brevinin-1TYa (orange). Twofold serially diluted peptide solutions were incubated with ABTS⁺ in a U-bottom 96-well microplate at 25 °C for 10 min. After incubation, the A₇₃₄ values were measured and the inhibition percentage of ABTS was calculated. Points and vertical bars represent the mean ± SE (n = 4). Values with the same letters are not significantly different (p ≥ 0.05). Neither brevinin-1TYa nor granuliberin-SSa (black; negative control) [26] exhibited any antioxidant activity.

Figure 8

(A) Evaluation of cytotoxic effects of yakushimin-TYa (blue), amurin-9TYa (green), and brevinin-1TYa (orange) in eukaryotic cells. Media containing 5 × 10³ COS7 cells or 1 × 10⁴ HUVECs in 100 μL aliquots were incubated with twofold serially diluted peptides for 2 h at 37 °C. Cell proliferation was determined using an MTT assay, and the survival rates were expressed relative to the control (0 dose). Columns and vertical bars represent the mean ± SE (n = 4). Values with the same superscripts are not significantly different (p ≥ 0.05). (B) SEM analysis of COS7 cells and HUVECs treated with yakushimin-TYa (128 μg/mL), amurin-9TYa (64 μg/mL), brevinin-1TYa (128 μg/mL), or myoglobin (128 μg/mL, negative control) for 1 h at 25 °C. Abnormally shaped cells, such as those showing cell shrinkage, surface destruction (arrows), or nucleus destruction (arrowhead), are visible in all panels except in the amurin-9TYa-treated HUVECs, compared to the myoglobin-treated cells. Scale bar = 20 μm.