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

摘要

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 ℃ for 30 min, 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 H_2O_2 and other reactive components since it was not counteracted by H_2O_2 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.