Abstract
Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.
Highlights
Anuran skin is source of a large variety of substances with different biological activities, such as biogenic amines, steroids, alkaloids, bufadienolides, peptides, and proteins [1]
Our results suggest that the highest activity observed in the presence of NaCl or CHAPS was achieved due to the increase of the ionic strength of the incubation buffer
Concluding Remarks The peptidases present in the crude skin secretion of the pepper frog Leptodactylus labyrinthicus have different specificities or are broad-specificity peptidases. It was not found in the skin secretion of Leptodactylus truncated peptides derived from hydrolysis between the positions 1 and 11
Summary
Anuran skin is source of a large variety of substances with different biological activities, such as biogenic amines, steroids, alkaloids, bufadienolides, peptides, and proteins [1]. Most of these compounds are produced by the granular glands present in the skin of the dorsal region and are involved in protecting against predators and pathogens [2,3]. Antimicrobial peptides (AMPs) show an important role in innate immunity, besides being important to angiogenesis, tegument repair, inflammatory processes, and chemotaxis [4]. Similar peptides are present in both secretions and tissues due to the common embryonic-ectodermal origin of the vertebrates’ skin and brain [5,6,7]
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