Fig 1.
Nucleotide sequence of callitide.
(A) Nucleotide and translated amino acid sequence of cloned callitide biosynthetic precursor-encoding cDNA. The putative signal peptide is double-underlined, and the mature callitide sequence is single-underlined. The stop codon is indicated with an asterisk; (B) Alignment comparison of callitide and sauvatide precursor. (1) Putative signal peptide. (2) Acidic spacer peptide-1. (3/5) Propeptide convertase processing sites. (4) Acidic spacer peptide-2. (6) Mature decapeptide. (7) C-terminal processing site with glycyl (G) residue amide donor indicated with an asterisk.
Fig 2.
Sepration and identification of callitide mature peptide.
(A) Region of reverse phase HPLC chromatogram of Agalychnis callidryas skin secretion. The arrow indicates the elution position of peak (#35) containing the myotropic decapeptide callitide; (B) LCQ mass spectrum of callitide from the skin secretion of Agalychnis callidryas. Ions at m/z 598.94 and 399.81 represent doubly charged (M+2H)2+ and triply charged (M+3H)3+ ions, respectively; (C) Electrospray ion-trap MS/MS fragmentation datasets derived from ions corresponding in molecular mass to callitide. Expected singly-, doubly- and triply- charged b-ion and y-ion fragment m/z ratios were predicted and observed fragment ions are indicated in bold type-face and are underlined; (D) MALDI-TOF mass spectrum of synthetic copy of callitide.
Fig 3.
(A) Dose-response curve of synthetic callitide using rat urinary bladder smooth muscle preparations. Each point represents the mean and standard error of six determinations. EC50 = 6.30×10−10 M; (B) Haemolysis of callitide on horse blood red cells.
Fig 4.
Predicted callitide structure and molecular docking.
(A) The 3D-model of callitide predicted by I-TASSER. The hydrophobic residues -ILV- in the middle section were colored in yellow while the two proline residues on the flank were colored in blue. (B, C) The validation of the predicted callitide (B) and BKRB2 (C) models by z-score using ProSA; (D) The peptide callitide cluster in complex with BKRB2. The C-terminus the peptides were inserted into BKRB2, and the peptides were colored; (E) The predicted interactions of callitide-BKRB2. Callitide and the receptor residues predicted to be involved in ligand binding were shown as sticks and labeled. Callitide backbone was colored by green with oxygen atoms colored by red and nitrogen atoms colored by blue. The related residues in BKRB2 were colored as cyan, and yellow dot lines indicated the interactions.
Table 1.
Alignments of mature peptide sequences and origin species.