Identification of New Ocellatin Antimicrobial Peptides by cDNA Precursor Cloning in the Frame of This Family of Intriguing Peptides

Abstract

Ocellatins are a family of antimicrobial peptides found exclusively in the Leptodactylus genus. To date, 10 species have been studied and more than 23 peptides described. Here we report the sequences of five new peptides from the skin of the frog Leptodactylus latrans (Anura: Leptodactylidae) determined by cDNA cloning of the complete prepro-peptide structures. The mature peptides were characterized with in silico tools and compared with those previously described. With 21 amino acid residues, this new set of peptides not previously described in the Leptodactylus genus share between 100 and 76.2% similarity to ocellatin antimicrobial peptides. These novel peptides are cationic and their three-dimensional (3D) structure holds the highly conserved residues G¹, D⁴, K⁷, and K¹¹ and a high theoretical amphipathic α-helix content. Furthermore, in silico analyses of these new peptides predicted antimicrobial activity. This study is framed in the context of previous work published about ocellatins, and therefore, provides a review of this intriguing family of peptides.

Keywords: Leptodactylus latrans; bioprospection; in silico techniques; leptodactylidae; natural products.

Figure 1

Deduced amino acid sequences of the ocellatin precursors of Leptodactylus latrans. Prepro-regions (signal peptide and acidic region boxed white and gray respectively) and a variable domain (boxed black) that correspond to mature peptide are indicated. Cys residue indicates the end of the signal peptide and the Lys–Arg processing site shows the end of the acidic region.

Figure 2

Sequence motifs common to the 28 ocellatin family peptides described to date. The amino acids are stacked on top of each other in increasing order of frequency and plotted. The height of each letter is proportional to the observed frequency of the corresponding amino acid. The overall height of each stack is proportional to the sequence conservation at that position. Amino acid color code: green = polar (G, S, T, Y, C, Q, N), blue = basic (K, R, H), red = acidic (D, E), and black = hydrophobic (A, V, L, I, P, W, F, M).

Figure 3

Cladogram of all ocellatin peptides identified from the Leptodactylus genus to display similarity, and the associated anuran species in which they were identified (following the color code).

Figure 4

Physical-chemical properties of ocellatins. Net charge, hydrophobicity (GRAVY), amphipathicity (µH), and percentage of α-helix content.

Figure 5

Three-dimensional (3D) structure prediction, hydrophobic, and hydrophilic faces and Schiffer and Edmundson helical wheel projection diagrams of the peptides identified from the skin secretion of Leptodactylus latrans. Diagrams show α-helix motifs with amphiphilic peptide structures having a hydrophobic (red region) and hydrophilic region (blue region). Amino acid color code for the wheel projections: yellow = non-polar/hydrophobic (Leu, Val), gray = Gly, blue = basic (Lys, Arg), purple = polar without charge (Thr), pink = polar without charge (Asn), and green = Pro.