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.

Keywords: Agalychnis callidryas; anticancer effect; bradykinin antagonist; myotropic activities; proline-rich peptide.

Figure 1

(A) Nucleotide sequence of frog skin-derived cDNA encoding the biosynthetic precursor of AcT-3. The putative signal peptide marked with double-underline, mature peptide marked with single-underline and stop codon (asterisk) is indicated. (B) Alignments of open-reading frame amino acid sequences of AcT-3 and PhT-3. The main structure of the precursors is indicated. (1) Putative signal peptide. (2) Acidic spacer peptide. (3) Mature peptide. (4) C-terminal processing site. Conserved residues are indicated by asterisks.

Figure 2

(A) Reversed phase HPLC separation of skin secretion components from Agalychins callidryas. The absorbance was monitored at 214nm. The retention times of AcT-3 was indicated by arrows. (B)MALDI-TOF mass spectrogram of AcT-3 (m/z=1538.19) from a reverse phase HPLC fraction of the frog skin secretion.

Figure 3

(A) Solarix FT-ICR-MS full scan of AcT-3 from reverse phase HPLC fraction. The doubly charges ion (m/z 769.88), triply charged ion (m/z 526.24) of a peptide with parent mass of 1539.79Da were detected. (B) MS/MS fragmentation spectrum of doubly charged ion (m/z 769.88).

Figure 4

(A) Dose-response curves of Bradykinin (●) and AcT-3 (■) in rat tail artery smooth muscle preparations. (B) Dose response of BK in the rat tail artery smooth muscle (●) and BK dose response in AcT-3 pre-incubated (10^-6M) artery smooth muscle (■). (C) Dose-response curves of AcT-3 (●) in rat small intestinal smooth muscle preparations. Error bars indicate the mean±SEM. Six replicates of each point were recorded.

Figure 5

AcT-3 inhibits the proliferation of androgen-dependent prostate cancer LNCaP cells and androgen-independent prostate cancer PC3 and DU145 cells in 72h. LNCaP cells were treated with AcT-3 and incubated for 24h (A), 48h (B) and 72h (C). PC3 cells were treated with AcT-3 and incubated for 24h (D), 48h (E) and 72h (F). DU145 cells were treated with AcT-3 and incubated for 24h (G), 48h (H) and 72h (I). Statistical analysis was performed using Student's t-test. Data shown represents mean±SEM of three separate experiments. Six replicates were recorded in each experiment. Asterisks indicate *p<0.05, **p<0.01, ***p<0.001 versus controls.

Figure 6

Detection of DNA fragmentation in the cell cytoplasm of human prostate cancer cell lines (A: LNCap; B: PC3; C: DU145) treated with AcT-3. Three replicates at each point were recorded.

Figure 7

Haemolytic effect of AcT-3. Three replicates at each point were recorded. (Positive control: 2% red cell suspension + equal volume PBS containing 2% Triton-100; Negative control: 2% red cell suspension + equal volume PBS).