Unravelling skin secretion peptidomes of Cruziohyla calcarifer, Agalychnis spurrelli and Hypsiboas picturatus (HYLIDAE)

This thesis describes the structural and functional characterization of the skin secretion peptidomes from Cruziohyla calcarifer, Agalychnis spurrelli and Hypsiboas picturatus. Firstly, the peptidome of C. calcarifer contained 53 peptides arranged in 11 peptide families including: 7 Kazal-type proteinase inhibitors (with 18 variants), 18 cruzioseptins (novel antimicrobial peptide family), medusin-CC, [Ser6,lle10,Tyr11 ]-phyllokinin, phylloseptin-CC1, plasticin-CC1/CC2, dermaseptin-CC1/CC2, tryptophyllin CcT2/CcT2-2, 1 proline-arginine rich peptide, 3 long alpha helical peptides, and 4 orphan peptides. Secondly, the peptidome of A. spurrelli contained 13 peptides arranged in 6 peptide families: [Ser6,Val10,Asp11 ]-phyIlokinin, [Thr6,Val10,Asp11 ]-phyllokinin, medusin-AS, tryptophyllin-AsT 1 /AsT3, dermaseptin-SP2 to SP5, phylloseptin-SP1, and 3 orphan peptides. Thirdly, the peptidome of H. picturatus contained 7 novel peptides: 3 picturins (25-mer) and 3 pictuseptins (22-26-mer) -both novel families- and a 55-mer peptide of alpha helical structure. The physicochemical properties of these 7 novel peptides suggested antimicrobial activity. Phyllogenetic analysis performed with the precursor sequences of these peptides indicated that most of them did not cluster by species, but instead they clustered by peptide type. In addition, analysis by tandem mass spectrometry found co-occurrence of the same peptide in two or three species and such peptides included protease inhibitors and antimicrobials. Overall, these data support a common genetic origin, where the ancestral gene has undergone a series of gene duplications with subsequent divergence of loci, which under diversifying selection has produced the current hypervariable peptides. Finally, the biological roles of antimicrobial/cytolytic peptides, myotropic peptides, Kazal-type protease inhibitors and alpha long helical peptides were predicted through bioinformatic comparisons. It is proposed that the Kazal-type protease inhibitors have a regulatory role, the alpha long peptides may be involved in peptides and nucleic acid packing in secretory granules and cytolytic peptides -below their lytic concentrations- may permeabilize cellular membranes assisting delivery of other small pharmacologically active peptides to their intracellular targets.