Papel de proteínas na tinta liberada pela lesma-do-mar Aplysia dactylomela Rang 1828 os mecanismos de defesa do animal

Abstract

The sea hare Aplysia dactylomela is known by discharging a purple ink when disturbed. As it doesn't show any external structure of protection, it is believed that this secretion, rich in bioactive substances, participates in the chemical defense against pathogens and/or predators. The ink is constituted mainly by pigments, proteins and low molecular mass substances. The pigments are originated from red algae, nevertheless, there is lack of information about the origin, processing, storage and function of the ink proteins. This work describes the protein composition of the ink and present some physiochemical and biological characteristics of the dactylomelin-P, an antibacterial protein from the ink besides information concerning the location of this protein in the purple gland. The ink was obtained from specimens collected at Fleixeiras beach, Ce. The protein composition of the ink was studied by bi-dimensional electrophoresis. The purification of dactylomelin-P consisted basically in ion exchange and hydrophobic interaction chromatography. Dactylomelina-P was analyzed as to molecular mass, isoeletric point, amino acid composition, carbohydrates, heat stability, pH and proteinase resistance. Several biological activities were tested with the ink and with dactylomelina-P, including antimicrobial, enzymatic, haemagglutinating, anticoagulant, hemolytic, cytotoxic and toxic activities. The immunolocalization assays of dactylomelina-P were carried out in different sea hare tissues by western blot and immunohistochemistry and the interaction with Staphylococcus aureus was investigated by immunocytochemistry. The ink of A. dactylomela contains more than 40 proteins/peptides, with molecular masses below 70 kDa and acid pIs. The most abundant protein in the ink is the dactylomelin-P, a monomeric protein with 59,8 kDa, pI 5,0, high methionin content, and less than 1% carbohydrates. It is denatured at 60 ºC for 10 minutes and it resists to pH range of 3-12. Dactylomelin-P shows a wide antibacterial action spectrum, but no antifungal, unlike the ink that possesses some factor with this activity. It is particularly efficient against sea bacteria, can be bactericidal (4,0μg/ml) or bacteriostatic (0,2μg/ml), depending on the concentration. Dactylomelin-P agglutinated rabbit, mice and rat erythrocytes, but it did not show anticoagulating, hemolytic and cytotoxic activities. The LD50 for mice was 60-100 mg/Kg, being considered moderately toxic. Dactylomelin-P was only found in the ink gland, being located preferentially in the cells of the producing vesicles. The electronic transmission microscopy revealed that the dactylomelina-P crosses the cell wall of S. aureus and interacts mainly with the plasmatic membrane, probably interfering in the metabolism of the bacterium, instead of causing mechanic damage to the cell.