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

<b>Background/Objective:</b> 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 <i>Rana tagoi yakushimensis</i> and analyze their functions. <b>Methods:</b> 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. <b>Results:</b> 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 <i>Staphylococcus aureus</i>, and brevinin-1TYa and amurin-9TYa induced morphological changes in <i>Escherichia coli</i> and <i>S. aureus</i>. Yakushimin-TYa, amurin-9TYa, and brevinin-1TYa showed concentration-dependent antibacterial effects against the plant pathogens <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> and <i>Clavibacter michiganensis</i> subsp. <i>michiganensis</i>. 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. <b>Conclusions:</b> These findings highlight the potential of these peptides, particularly amurin-9TYa, for future applications as antimicrobial and therapeutic agents.