Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry. Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified. Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A.
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Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry.
Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified.
Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A. The resistance of microorganisms to current chemotherapy has limited successful treatments which stresses the urgent need for more efficient therapeutic strategies [ 1 ].
Animal venoms and secretions, such as the skin secretions of frogs, represent a treasure trove of small molecules that offer a promising way to tackle this global challenge [ 2 ]. The skin secretions of phyllomedusine hylids are complex molecular libraries composed by alkaloids, biogenic amides, inorganic molecules, opioids, steroids, nucleic acids, and proteins, and are mainly rich in a promising array of diverse peptides [ 3 , 4 ].
During the last 30 years, a large number of studies have reported the isolation, and biochemical and pharmacological characterization of frog peptides. Nearly peptides have been described from 19 of 60 species of Phyllomedusinae. The most studied genus is Phyllomedusa , for which 13 species have revealed peptides [ 4 ]. The next is Agalychnis, with five species analysed and 33 peptides identified; followed by Cruziohyla, with 33 peptides identified in one species; and Phasmahyla, for which the only species studied has revealed 23 peptides [ 5 , 6 , 7 , 8 ].
A comprehensive study of the skin secretion peptides of Agalychnis spurrelli , a frog found from southern Costa Rica to central-western Ecuador, remains to be performed. From the structural point of view, these peptides showed a valuable chemical diversity of molecular architectures, from short molecules with few amino acids and a simple secondary structural element, such as temporins [ 9 ], to protease inhibitor peptides with tridimensional structures more complex and classified in several types [ 5 ].
Consequently, these variable scaffolds render them versatile compounds and potentially useful for the treatment of several diseases [ 5 ]. Peptides from frog skin present a wide repertoire of biological activities; for example, dermaseptins B2 and B3 have antitumor potential [ 10 ], [D4k]ascaphin-8 is anti-inflammatory candidate [ 11 ], and magainin-AM2 has been proposed for the diabetes therapy [ 12 ].
Representatives of several peptide families have been reported from Agalychnis skin secretions, such as dermaseptins, phylloseptins, and medusins. These peptides have a broad spectrum of cytolytic activity; i. DRS-L1 showed high potency against E. In addition, DRS-L1 was selectively cytolytic against red blood cells in contrast to HepG2 cells—a human liver cancer cell line [ 20 ]. Phylloseptins are antimicrobial peptides that contain the sequence FLS at their N-terminals, an H His in position 7, and C-terminal amidation.
In Agalychnis , three medusins have been reported: medusin AC of A. Both medusins were active against Staphylococcus aureus and Candida albicans , without a haemolytic effect at their minimum inhibitory concentrations MICs [ 22 ]. Moreover, medusin-AL same sequence as medusin-AC was active against the chytrid fungus Batrachochytrium dendrobatidis , and it showed a similar cytolytic activity against erythrocytes and HepG2 cells at its lowest concentrations [ 20 ]. There have been significant advances in peptide sequencing, brought about by combining the techniques of tandem mass spectrometry with data-dependent acquisition and molecular cloning which generates the peptide databases.
In addition, the ability to prepare cDNA libraries from RNA isolated from skin secretions, rather than from the skin glands themselves, is a less invasive approach [ 23 , 24 , 25 ]. Integrating these biochemical techniques has helped with gaining deeper insights into the spectrum of peptides in complex samples [ 19 , 26 , 27 , 28 ].
In this context, this work aimed to identify and characterize the primary structures of peptides from the skin secretion of Agalychnis spurrelli by integrating proteomic and molecular tools, and subsequently, to evaluate their antimicrobial activity. Six specimens of Agalychnis spurrelli were employed in this study: two wild adults, collected in northwestern Ecuador and four captive-bred juveniles, provided by Centro Jambatu for Research and Conservation of Amphibians, in Ecuador.
The wild frogs were returned to their habitat after the extraction and captive frogs were returned to their enclosures. Five milligrams of freeze-dried skin secretion of A.
Fractions 1 mL were collected each minute. Absorbance at nm and nm was monitored continuously. Twenty microliters of the remaining skin secretion samples, dissolved in buffer A from the HPLC step, were subjected directly to an analytical column Phenomenex C; 4.
Nucleotide sequences were analysed by MEGA 6. Signal peptides were predicted using the SignalP 4. Secondary structure prediction was performed using the SOPMA programme and the physicochemical properties of the peptides were calculated using HeliQuest Compu Parameters and the Peptide property calculator from Bachem [ 34 , 35 , 36 ].
For the in vitro evaluation of antimicrobial potentials of new biochemical structures identified herein, five synthetic peptides were purchased from a commercial supplier Biomatik. Approximately mg of each peptide was synthetized by Fmoc technology. Minimal inhibitory concentration of the synthetic peptides was determined against the following Gram-positive bacteria: Staphylococcus aureus ATCC and S. Seven replicates per peptide concentration was employed and experiments were performed in triplicates.
Aiming to determine the toxicity of frog peptides to red blood cells, in vitro assays were performed with different concentrations of each peptide. After the incubation period, the cells were centrifuged at rpm for 5 min and transferred to a 96 well plates. The haemolytic activity was monitored at nm and was performed in triplicate.
Eleven nucleotide sequences encoding peptide precursors were successfully cloned from the skin secretion of Agalychnis spurrelli. Among these were two phyllokinins and one medusin that were all reported previously in other species.
The other eight mature peptide sequences were novel, including one phylloseptin, four dermaseptins, and three unclassified peptides.
The fractions containing dermaseptin-SP2 and phylloseptin-SP1 are identified with the letters A and B in the chromatogram, respectively. Herein we describe the main findings of each peptide family, with emphasis on novel primary structures of dermaseptins and phylloseptin, identified in this sample.
Detection at nm red line ; detection at nm green line. The translated open reading frames consisted of — nucleotides that encoded: 1 a 22 amino acid putative signal peptide; 2 a 29 amino acid acidic spacer that contained two pro-peptide processing sites KR and a sequence of four amino acids ESPD or ESPE at the C-terminal side of the latter; and 3 an 11 amino acid mature peptide Supplementary Figure S1.
These phyllokinin were named [Ser 6 , Val 10 , Asp 11 ]-phyllokinin and [Thr 6 , Val 10 , Asp 11 ]-phyllokinin to highlight their similar but different amino acid sequences. The open reading frame consisted of: 1 a 22 amino acid putative signal peptide; 2 a 27 amino acid acidic spacer that contained two KR pro-peptide processing sites; 3 an 18 amino acid mature medusin sequence; and 4 a glycine amide donor residue Supplementary Figure S2.
This peptide precursor was named medusin-AS to represent its origin in A. The translated open reading frame consisted of: 1 a 22 amino acid putative signal peptide; 2 a 23 amino acid acidic spacer with one pro-peptide processing site; 3 a 22 amino acid mature peptide; and 4 a glycine residue amide donor at the C-terminus Figure 2 and Supplementary Figure S3.
Precursor domain structures: 1 Putative signal peptides. Comparison of amino acid sequence similarity of phylloseptins. The translated open reading frames consisted of: 1 22 amino acid putative signal peptides; 2 23 amino acid acidic spacers that contained two pro-peptide processing sites KR and RR; 3 27—28 amino acid mature peptide sequences; and 4 a tripeptide sequence GEQ at the C-terminus, where glycine acts as an amide donor Figure 2 and Supplementary Figure S3.
Comparison of amino acid sequence similarity of dermaseptins. Its monoisotopic molecular mass of The fraction also contained another peptide of However, its translated mature sequence was highly divergent and did not produce any significant hits when compared with the databases in NCBI.
The peptide database created by molecular cloning was compared with the database generated by fragmentation of the peptide ions in the tandem mass spectrometric analysis of the skin secretion of A. In summary, 15 to peptide fragments were sequenced per peptide, which contained between 11 and 31 amino acids and a molecular mass range of between The synthetic dermaseptins showed antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeast. The peptides were more active against bacteria than yeast Table 2.
The dermaseptin-2 was the most potent peptide, inhibiting bacterial growth at 2. Dermaseptins induced no red blood cell lysis at MIC concentrations. The phylloseptin-SP1 showed greater toxicity to red blood cells than did the dermaseptins, also increasing with increasing peptide concentration. In addition, helical wheel plots illustrate the amphipathic characteristics and properties of alpha helices in the analysed peptides Figure 6.
Helical wheel projections of the phylloseptins and dermaseptins-SP of A. The granular glands of frogs are responsible for the production of diverse peptides, some of which have shown a broad spectrum of action with potential biomedical applications [ 2 ]. Other biochemical studies have revealed similar molecular complexity and diversity from skin secretions of frogs [ 4 ]. By peptidomic analysis and integrating tandem mass spectrometry with molecular cloning, eleven peptides belonging to five families were identified in A.
The peptide sequences identified in this work are detailed and discussed in the light of their families. The deduced amino acid sequences revealed the presence of two previously reported bradykinin-like peptides. The nucleotide comparison of frog peptides precursors-encoding cDNA, ignoring the vespakinin-T precursor because it is too divergent, revealed that these related phyllokinin precursors have 24 non-synonymous substitutions that encode for 12 amino acids and eight synonymous substitutions in their signal and acidic spacer peptides.
In addition, there are eight synonymous substitutions in the region corresponding to the mature peptides between these precursors. The occurrence of the same Thr 6 -phyllokinin in the three species of Agalychnis is not surprising and reflects taxonomic relationships. In contrast, the presence of the bradykinin-like peptide RD of A. Ascaphus truei is an ancient basal species and sister-group of all other amphibian species. Therefore, this could show an early evolutionary origin of the ancestral gene of this bradykinin-like peptide, which should be confirmed whenever the precursor sequence of RD becomes available Supplementary Table S1 [ 43 ].
Here, we have named this peptide precursor as [Ser 6 , Val 10 , Asp 11 ]-phyllokinin for the sake of consistency with other bradykinin-related peptides of phyllomedusine frogs. The occurrence of this bradykinin-like peptide vespakinin-T in the venom of the hornet, V. A previous study showed that RD is able to induce relaxation of pre-contracted mouse trachea interacting with the bradykinin B2 receptor in the same way as bradykinin, only less potently [ 43 , 44 ].
Although the biological role of these bradykinin-related peptides remains unclear, the observation of antipredator behaviour in some snakes, after the initial taste of some frogs that contain these peptides, indicates a defensive role.
In addition, studies that show bradykinin to be a potent stimulator of gastrointestinal smooth muscle and pain sensitivity pathways in mammals, as well as its effect on the vascular system of snakes, strongly suggest an antipredator-defensive role among these peptides [ 3 , 45 , 46 ]. A medusin with an identical amino acid sequence to medusin-AC was identified in this skin secretion.
The nucleotide sequences of corresponding precursors are virtually identical, having three non-synonymous substitutions that lead to three different amino acids and four synonymous substitutions in the region corresponding to the signal peptide and acidic spacer.
In addition, the mature peptide sequence contains only one synonymous substitution. It is notable that this medusin is highly conserved across species. At the moment, medusin-AC is known to occur in A. Supplementary Table S2. This is the first antimicrobial peptide occurring in four different species of two distinct genera and could be indicative of a secondary role acting on cell receptors.
At the moment, this peptide has been found to have antimicrobial activity against S. A novel phylloseptin was identified in the skin secretion of A. As shown in our findings, conserved amino acids are concentrated in their N-terminal regions, while the C-terminal end is highly divergent.
These are all the main characteristics of phylloseptins, so the new sequence was named phylloseptin AS-1 in accordance with the nomenclature proposed for antimicrobial peptides of this family [ 38 ].
Gliding tree frog
The extensive webbing on the hands and feet of Agalychnis spurrelli can act as a parachute, helping slow and lengthen their descent Scott and Starrett For an excellent picture of this behavior, see Savage Species description based on Ibanez et al , Duellman and Savage Large treefrog. Males to Individuals from Panama are much larger than individuals from Costa Rica.
Agalychnis spurrelli (Captive bred Gliding Tree Frog) - Sub-Adult
It is a nocturnal tree frog species of tropical rainforests. It moves into the canopy especially in the dry season climbing branches and vines, although it generally uses vertical substrates available in the habitat. Frogs glide to skydive. While gliding they extend legs and hands so that they can be seen throughout its length.
The gliding tree frog Agalychnis spurrelli is a species of frog in family Phyllomedusidae. Other common names are the gliding leaf frog , Spurrell's leaf frog , and pink-sided tree frog. Its natural habitats are subtropical or tropical moist lowland forests and intermittent freshwater marshes. It is threatened by habitat loss. The head is broad and the eyes are large, with reticulated lower eyelids.