Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Abstract

Background: Argenteohyla siemersi (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. Methods: The A. siemersi 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 2 , clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi 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 2 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. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.

Variation of Protein Band Pattern on the Skin Secretions of Chalcorana chalconota (Schlegel, 1837) Complex

“…The parotoid gland secretion of the R. marina toad has seven protein bands 9 . Argenteohyla siemersi frog skin secretions had six protein bands 10 .…”

“…Fusco et al 10 reported that the secretion of the skin frog Argenteohyla siemersi showed six protein band patterns with molecular weights ranging from 15-55 kDa. Based on existing research on several Amphibian species, it can be estimated that there are variations in the protein band patterns on the skin secretions of C. parvaccola and C. rufipes, which are part of the C. chalconota species complex.…”

Neglected Venomous Animals and Toxins: Underrated Biotechnological Tools in Drug Development

“…Enzymes presenting such activities have already been described as important toxins for snake venoms [81,82] and were also described in some amphibians from different genus, for example phospholipase in Pithecopus azureus [83] and serine peptidases in Duttaphrynus melanostictus [84]. Furthermore, Fusco et al 2020 [85] studied the epidermal secretion of Argenteohyla siemersi and described both phospholipasic and hemolytic activities. They also reported that that venom is cytotoxic and capable of promoting necrosis which is independent of the proteolytic activity, a different activity pattern from C. greeningi (included in the same genus).…”