Fusco Luciano S, Cajade Rodrigo, Piñeiro Jose M, Torres Ana M, da Silva Igor R F, Hyslop Stephen, Leiva Laura C, Pimenta Daniel C, Bustillo Soledad
Protein Research Laboratory (LabInPro), IQUIBA-NEA CONICET, National University of the Northeast, Corrientes, Argentina.
Herpetology Laboratory, National University of the Northeast, Corrientes, Argentina.
J Venom Anim Toxins Incl Trop Dis. 2020 Mar 30;26:e20190078. doi: 10.1590/1678-9199-JVATITD-2019-0078.
(red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied.
The skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line.
The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis.
Results presented herein evidence the biochemical composition and biological effects of skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.
红斑阿根廷树蛙是雨蛙科的一种有盔头的树蛙物种。该物种具有复杂的反捕食防御机制组合，其中包括一种具有高度致死性的皮肤分泌物。然而，这种分泌物的生化组成和生物学效应尚未得到研究。
通过质谱和色谱分析（基质辅助激光解吸电离飞行时间质谱、反相高效液相色谱和气相色谱 - 质谱）对皮肤分泌物样本进行分析。还通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳研究蛋白质。在评估的生物学活性中，评估了几种酶活性（溶血、磷脂酶A、凝血、蛋白水解和酰胺水解）。此外，对C2C12细胞系的成肌细胞评估了细胞毒性活性（细胞溶解和荧光染色）。
基质辅助激光解吸电离飞行时间质谱分析鉴定出皮肤分泌物水溶液中的多肽和蛋白质。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示存在分子量为15至55 kDa的蛋白质。使用质谱检测到类固醇，但未检测到生物碱或肽（小于5 kDa）。通过气相色谱 - 质谱分析，皮肤分泌物的甲醇提取物中显示存在脂质。这种分泌物表现出溶血和磷脂酶A活性，但没有酰胺水解、蛋白水解或凝血活性。此外，证明了皮肤分泌物对培养的C2C12成肌细胞具有剂量依赖性细胞毒性。形态学分析、乳酸脱氢酶释放定量和荧光染色表明，这种分泌物引发的细胞死亡涉及坏死。
本文给出的结果证明了皮肤分泌物的生化组成和生物学效应，并有助于了解有盔头树蛙的防御机制。
