Hyposeptins: A Novel Family of Histone-derived antimicrobial peptides from the skin secretion of Phyllomedusa hypochondrialis.

Brand, G.D.¹,2,4, Leite, J.R.S.A.¹,3,4, Bloch Jr., C.¹

1 Mass Spectrometry Lab., EMBRAPA - Genetic Resources and Biotechnology; 2 Department of Physiology, Institute of Biology, University of Brasilia, Brasília - DF, CEP 70910-900, Brazil; 3 Department of Cell Biology, Institute of Biology, University of Brasilia; 4 SABIN Clinical Analysis Laboratory, Brasília – DF, Brazil.

Introduction: Even though the skin secretions of frogs belonging to the Phyllomedusinae subfamily have been researched for more than 3 decades, their full richness is only beginning to be unveiled. The progress in biochemical methods witnessed by the last few years now allow the characterization of molecules present in previously undetectable amounts. This is surely the case of Hyposeptins 01 and 02, which are histone-derived antimicrobial molecules from Phyllomedusahypochondrialis, a tree-frog from the Cerrado region of Brazil. This work describes their isolation, molecular mass determination as well as de-novo MS/MS sequencing, along with the mass spectrometric determination of post-translational modifications. Material and Methods: The liophilized extract obtained from the skin by mild shock was purified by RP-HPLC (semi-prep and analytic columns from Vydac C18), analyzed in MS and MS/MS modes in a Ultima Q-ToF API (Micromass) and ABI 4700 ToF/ToF (Applied Biosystems) Mass Spectrometers. Similarity searches were performed under Expasy Molecular Biology server (www.expasy.org). Results: Hyposeptin 01 and Hyposeptin 02 are [M+H]⁺=1519,01 and [M+H]⁺=12450,01 Da peptides. MS/MS de-novo Sequencing revealed that Hyposeptin 01 is a 14-residue N-terminally acetylated and C-terminally amidated peptide, and also that Hyposeptin 02 is a truncated form of the first without the two N-terminal aminoacids. Similarity searches revealed that both molecules have high similarity to the Histone H1 Late Precursor from Sea Urchin. Conclusion: Although data on the antimicrobial potency and synergistical relations of Hyposeptins to other peptides have not been taken yet, the identification of a novel family adds further possibilities for the creation of novel antimicrobial agents capable of treating diseases caused by multi-resistant microorganisms.

Supported by: SABIN, CAPES, CNPq, EMBRAPA – Genetic Resources and Biotechnology.

Correspondence to: gdbrand@terra.com.br