The Crude Skin Secretion of the Pepper Frog Leptodactylus labyrinthicus Is Rich in Metallo and Serine Peptidases

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.

Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions

“…It is known that fragments derived from active peptides can be found in the skin secretion of frogs and, in particular, it was shown that the crude skin secretion of Leptodactylus labyrinthicus is rich in metallo and serine peptidases [49]. The peptide sequences investigated here present 100% homology from residues 1 to 22 (ocellatin-LB1 primary structure), ocellatin-LB2 carries an extra Asn and ocellatin-F1 extra Asn-Lys-Leu residues.…”

Systematics of the Neotropical GenusLeptodactylusFitzinger, 1826 (Anura: Leptodactylidae): Phylogeny, the Relevance of Non-molecular Evidence, and Species Accounts

“…fallax, Krintler, 1986), and the dermal glands of several species are known to produce defensive skin secretions (Cei et al, 1967), including antimicrobial peptides and other protein toxins found in L. fallax (Rollins-Smith et al, 2005;King et al, 2005a), L. labyrinthicus (Libério et al, 2014), L. laticeps (Conlon et al, 2009), L. latrans (Nascimento et al, 2004(Nascimento et al, , 2007Leite et al, 2010), L. pentadactylus (Habermehl, 1981;Barlow, 1998;King et al, 2005b;Limaverde et al, 2009;Sousa et al, 2009), L. syphax (Dourado et al, 2007). Similarly, the foam nests of L. vastus possess a novel surfactant protein (Hissa et al, 2008(Hissa et al, , 2014.…”

Animal–microbe interactions and the evolution of nervous systems

“…The skin of frogs also contains many anti-microbial peptides [125], including several FLPs [126]. Finally, frog skin also contains numerous peptidases [127]. Thus, it seems that unless the nervous system were internal to the epidermis, the multiple FLPs in both neurons and skin cells, multiple FLP receptors on neurons, and multiple peptidases produced by skin cells could all interfere with both neural signalling and epithelial-microbial signalling in frogs.…”