Brevinin-1E

Skin secretions of Rana saharica were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 36-43, 46-54 and 57-63 significantly stimulated insulin release by 2- to 8-fold compared with 5.6 mM glucose alone. Pooled fractions in the latter two bands were rechromatographed to reveal 9 homogenous peaks, which elicited significant 1.3- to 3.5-fold increases in insulin release (P < 0.05). Structural analysis of the most potent non-toxic peptides was performed by mass spectrometry and automated Edman degradation. This revealed four major insulin-releasing peaks with molecular masses of 2,676.9 Da, 3,519.3 Da, 4,920.4 Da and 4,801.2 Da respectively. These peptides were found to be identical to brevinin-1E, brevinin-2EC, esculentin-1 and esculentin-1B, which belong to the group of antimicrobial peptides isolated from skin secretions of various Rana frog species. Preliminary studies on the mechanism underlying the insulinotropic actions of esculentins-1 and -1B suggested possible involvement of both cyclic AMP-protein kinase A and -C-dependent G-protein sensitive pathways. These data indicate that the skin secretions of Rana saharica frogs contain bioactive molecules with significant insulin-releasing activity. Relatives of the brevinin/esculentin peptide family merit further investigation as novel insulin secretagogues.

Brevinin 1E, consisting of 24 amino acid residues, from Rana esculenta has potent antimicrobial and hemolytic activity. From a structural point of view, this peptide has a N-terminal hydrophobic region, a proline hinge region in the middle and a C-terminal loop region delineated by an intra-disulfide bridge, which is a common structural feature of antimicrobial peptides from Rana species. To investigate the structural features for antimicrobial and hemolytic activity, truncated and linearized brevinin 1E amides were synthesized and characterized. A deletion of three amino acids from the N-terminal region did not greatly affect antimicrobial activity but dramatically reduced hemolytic activity. The contribution of the intra-disulfide bridge to antimicrobial and hemolytic activity was somewhat different between brevinin 1E amide and truncated fragments. In brevinin 1E amide, the elimination of the intra-disulfide bridge did not greatly affect antimicrobial and hemolytic activity whereas the elimination of the intra-disulfide bridge in the truncated fragments did not decrease antimicrobial activity but did decrease hemolytic activity. Circular dichroism spectra and the retention time on the C18 reverse phase column revealed that the intra-disulfide bridge (i, i+6) formed an amphipathic loop which increased hydrophobicity and helped to induce the alpha-helical structure in the membrane-mimetic environment. Even though the intra-disulfide bridge and the N-terminal region were responsible for the alpha-helical structure and hydrophobicity, these two structural features were not essential for antimicrobial activity. The hemolytic activity of brevinin 1E amide and its analogs also correlated well with the retention time rather than the alpha-helicity.

Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC, is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide-bridged forms have relatively broad-spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKCAANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.