Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Abstract

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI=5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0–12.0), after heating to 55°C for 30min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H2O2 and other reactive components since it was not counteracted by H2O2 scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.