Poster 062 - Homotarsinin: A novel antimicrobial homodimer from Phyllomedusa tarsius skin secretion

Poster 62. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.421.

Homotarsinin: A novel antimicrobial homodimer from Phyllomedusa tarsius skin secretion.

1Prates, M.V.; 1Santos, N.C.F.; 1,2Leite, J.R.S.A.; 3Figueredo, R.C.R.; 4Martins, G.R.; 1Bloch Jr, C.

1 Laboratório de Espectrometria de Massa, EMBRAPA–Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil; 2 Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília-DF, Brazil; 3 Centro de Biologia Molecular Estrutural, LNLS, Campinas-SP, Brazil; 4 Laboratório SABIN de Análises Clínicas, Brasília, DF, Brazil.

In this work, we describe the isolation, purification and the complete N-terminal sequence a new peptide, named homotarsinin (Htr), isolated from the skin secretion of Phyllomedusa tarsius, a green tree-frog from the Phyllomedusinae subfamily, which inhabits the Amazonian basin, including Brazil, Colombia, Equator and Peru. The chromatographic profile revealed more than thirty fractions, from which the peptide (5505.18Da) was isolated, mass analyzed and purified using analytical RP-HPLC and MALDI-TOF/MS. Disulfide content investigation using in situ dithiothreitol reactions, followed by MALDI TOF-TOF/MS analysis, indicated the existence of a single S-S bond connecting two 2753.59Da polypeptide chains. After alkylation of the cysteine residues with iodoacetamide, the peptide was DeNovo sequenced by TOF/TOF-MS/MS and Q-TOF/MS/MS (CID fragmentation); Leucine, Isoleucine, and Lysine residues were confirmed by automatic Edman degradation, showing two identical polypeptide chains of 24 residues each.

Circular dichroism studies in different TFE solutions revealed that Htr assumes significant -helical content. MIC’s for Htr were determined against pathogenic E. coli, S. aureus and P. aeruginosa strains demonstrated the peptide in vitro bactericidal ability, with irrelevant litic effect against human erythrocytes. Previously, we have reported an antimicrobial heterodimer in P. distincta (Bastista et. al FEBS Lett. 2001; 494(1-2):85-9), with the present findings, it is possible to hypothesize about the existence of a new class of dimeric antimicrobial peptides in the skin secretion of the Phyllomedusa genus.

Support: Embrapa-Cenargen; Laboratório SABIN de Análises Clínicas; CNPq; FAPESP.