Proteomic Analysis of low molecular weight peptides from the skin secretions of Phylomedusa hypochondrialis.
Conceição, K.1, Konno, K.1, Melo, R.L.1, Antoniazzi, M.M.2; Jared, C.2, Lopes-Ferreira M.3 and Pimenta D.C.1
Laboratórios 1Especial de Toxinologia Aplicada – CAT/CEPID, 2Biologia Celular e 3Imunopatologia, Instituto Butantan, São Paulo, Brasil.
The amphibian skin secretions are sources of potential new drugs with medical and biotechnological applications, such as biogenic amines, complex alkaloids and peptides, which are produced by holocrine-type serous glands in the integument, where they are stored in the lumen of the cells and released upon stimulation. These molecules should play different roles, either in the regulation of physiological functions of the skin or in the defense against predators or microorganisms.
The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of this frog. In order to achieve this goal, crude skin secretion of Phyllomedusa hypocondrialis, a Hylidae that inhabits the Brazilian Caatinga, was pre-fractionated by solid phase extraction (SPE) and the peptides were separated by reversed-phase HPLC. First, the SPE-processed sample was fractionated into 29 pools. All the obtained fractions were monitored by MALDI-ToF mass spectrometry for evaluation of molecular masses of peptides and purification grade. These pools were screened for low molecular weight peptides (typically 700 to 1100 Da) with the aid of ESI-Q-TOF mass spectrometry. Several peptides were sequenced and three of them presented sequences that slightly resemble that of Bradykinin. These peptides have had their amino acid sequence determined by ‘de novo’ mass spectrometric sequencing and were identified as KPLWRL–NH2 (Phypo 3), RPLSWLPK (Phypo 5) and VPPKGVSM (Phypo 7a).
Bradykinin-like peptides were chosen for the direct administration of the crude skin secretion cause several effects on the circulatory system, including inflammation, as observed by intra-vital microscopy. These peptides are currently being synthesized for further investigation of their effect on isolated guinea-pig ileum and/or anesthetized rats.
Supported by CNPq, CAPES and FAPESP
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