Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

Abstract

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

“…However, when rat tail artery was pre-treated with RAP-L1, T6, L8-BK dose range from 1 × 10 −11 M to 1 × 10 −6 M followed by administration of BK at maximal effective concentration 10 −6 M as described previously [9], maximum inhibition of BK-induced relaxation was observed at 10 −6 M and 10 −5 M. Hence, we selected 10 −6 M as the most effective inhibitory concentration on rat tail artery smooth muscles (Figure 9). In the second series of experiments, when the rat tail artery smooth muscle preparations were treated with 10 −6 M RAP-L1, T6, L8-BK followed by the application of BK concentration range from 10 −11 M to 10 −5 M, the potency of BK-induced maximum relaxation of rat tail artery smooth muscle at 10 −9 M to 10 −5 M was dramatically decreased (nearly 40%–60%), although the IC 50 values were 1.20 ± 0.08 nM vs. 0.50 ± 0.56 nM.…”

Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire‐bellied toad, Bombina orientalis

“…MTT assay was used to determine the inhibitory effect of peptides on the proliferation of three human hepatoma cell lines (Hep G2, SK‐HEP‐1 and Huh7) and human prostate cancer cell line (PC3), human lung cancer line (A549) and human melanoma cell line (A375) as described by us previously . Briefly, cells were seeded in 96‐well plate at the density of 5 × 10 3 cells per well at 37°C for 24 hr.…”

Baltikinin: A New Myotropic Tryptophyllin-3 Peptide Isolated from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

“…This approach has been employed to assign biological roles and activities to a wide range of peptides from various species. In the meantime, the combination of functional genomics and structural data, which are produced from biosynthetic precursor cDNA cloning and functional target studies, can provide a holistic approach to the investigation of natural bioactive peptides [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].…”