Oncorhyncin III

Skin exudates of rainbow trout contain a potent 13.6 kDa anti-microbial protein which, from partial internal amino acid sequencing, peptide mass fingerprinting, matrix-associated laser desorption/ionization MS and amino acid analysis, seems to be histone H2A, acetylated at the N-terminus. The protein, purified to homogeneity by ion-exchange and reversed-phase chromatography, exhibits powerful anti-bacterial activity against Gram-positive bacteria, with minimal inhibitory concentrations in the submicromolar range. Kinetic analysis revealed that at a concentration of 0.3 microM all test bacteria lose viability after 30 min incubation. Weaker activity is also displayed against the yeast Saccharomyces cerevisiae. The protein is salt-sensitive and has no haemolytic activity towards trout erythrocytes at concentrations below 0.3 microM. Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels, indicating that its anti-bacterial activity is probably not due to pore-forming properties. This is the first report to show that, in addition to its classical function in the cell, histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH(2) by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content alpha-helices, which indicates that melectin can adopt an amphipathic alpha-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the alpha-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.

A potent antimicrobial peptide, tentatively named oncorhyncin II, was isolated from an acid extract of rainbow trout skin secretions. Amino acid sequencing showed that the first 17 residues of oncorhyncin II are identical to residues 138-154 of histone H1 from rainbow trout. Matrix-assisted laser desorption ionization mass spectrometry revealed that the purified peptide has a molecular mass of 7195.3Da. Taken together, these data indicate that oncorhyncin II is a 69-residue C-terminal fragment of histone H1, probably phosphorylated at two residues. Oncorhyncin II has minimal inhibitory concentrations in the submicromolar range against Gram-(+) as well as Gram-(-) bacteria and it does not display significant haemolytic activity towards trout erythrocytes. The purified peptide was found to induce a marked destabilisation of planar lipid bilayers without the formation of stable ion channels. Oncorhyncin II is possibly a cleavage product of histone H1 with a potentially important role in mucosal defence of rainbow trout.