Cleavage of Peptides from Amphibian Skin Revealed by Combining Analysis of Gland Secretion and in Situ MALDI Imaging Mass Spectrometry

Abstract

Peptides from skin secretions of amphibians are considered important components of their immune system and also play a relevant role in their defense mechanism against predators. Herein, by using mass spectrometry (MS), we characterize the sequence of 13 peptides from the gland secretion of the hylid tree frog, Boana punctata. Using in situ matrix-assisted laser desorption ionization imaging MS of a transverse section of the skin tissue, we show that some peptides are stored as longer molecules that are cleaved after being secreted, whereas others do not undergo any modification. Sequence comparison with peptides from other Boana species and analysis of the three-dimensional theoretical structure indicate that this cleavage depends on both the presence of a specific sequence motif and the secondary structure. The fact that peptides undergo a rapid cleavage upon secretion suggests that stored and secreted peptides may have distinct roles for anuran survival, including defense against pathogens and predators.

Figure 1

Amino acid sequence alignment of the 13 peptides identified in the gland secretion from dorsal skin of B. punctata and peptides described in other Cophomantini tree frogs. Arrow indicates the proteolytic cleavage site reported by de Magalhães and co-workers.

Figure 2

In situ MALDI IMS of dorsal (left panel) and lateral skin regions (right panel) of B. punctata. (A) Adult specimen (male). (B) Global MS spectra of detected ions. (C) Scan image of transverse sections. (D–G) MALDI-MS images reconstructed with ions m/z 2367.5 (D), 1976.2 (E), and 1683.3 (F) and colocalization of ions m/z 1683.3 and 2367.5 (G). (H) Light micrographs stained with hematoxylin and eosin. ep, epidermis; de, dermis.

Figure 3

Amino acid sequence alignment of three of the peptides identified in situ by MALDI IMS in the transverse section of dorsal and lateral skin samples of B. punctata (B. punctata_2367, B. punctata_1976, and B. punctata_1683) and similar or identical peptides identified in the gland secretion of B. punctata [B. punctata_P11, B. punctata_P12, and B. punctata_P13 (this article); B. punctata_Hsp9 and B. punctata_Psp10 and B. cinerascens_Crs]. Arrows indicate putative proteolytic cleavage site based on sequence identities between peptides identified by in situ MALDI IMS and peptides identified in the gland secretion.

Figure 4

3D structure prediction and Schiffer and Edmundson wheel projection diagrams of the peptides identified from the skin secretion of B. punctata. Diagram shows α-helix motifs with amphiphilic peptide structures having a hydrophobic and a hydrophilic region. Amino acid color code: yellow = unpolar/hydrophobic (Leu, Val), gray = Gly, blue = basic (Lys, Arg), purple = polar without charge (Thr), pink = polar without charge (Asn), and green = Pro.