First evidence of laccase activity in the Pacific oyster Crassostrea gigas

Abstract

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas.

First evidence of a potential antibacterial activity involving a laccase-type enzyme of the phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes

“…In a previous study, catecholase-type PO activity was detected in the haemolymph of C. gigas with L-DOPA and dopamine as substrates [13]. However, in the present study, dopamine displayed a toxic effect, while L-DOPA displayed no toxic effect on LGP32, when used alone at 1.25 mM concentration (data not shown).…”

“…In a previous study, laccase-type PO activity was detected in the haemolymph of C. gigas in the presence of the non phenolic substrate, PPD, but neither in the presence of the methoxy phenolic substrate, 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine), nor in the presence of the non phenolic substrate, 2,2 0 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) [13]. However, since PPD exerted an effect on bacterial growth in the absence of HLS in the present study (data not shown), results were expressed as the percentage of inhibition due to HLS (Table 1).…”

“…Catecholase-type PO activity was measured spectrophotometrically by recording the formation of o-quinones, by using the method described previously [13]. For all conditions, assays were performed with three 10-oyster pools.…”

Enzyme activity demonstrates multiple pathways of innate immunity in Indo-Pacific anthozoans

“…However, the presence of ultraviolet (UV) and visible-lightabsorbing melanin may be advantageous during times of temperature stress [8]. Laccase activity within anthozoans is therefore likely to be involved in clot stabilization [19] or another aspect of immunity, as hypothesized for the Pacific oyster, which also lacks an integument [22].…”

Towards an integrated network of coral immune mechanisms

“…While the tyrosinase-type melanin synthesis pathway has been the most intensively investigated with regard to invertebrate immunity [48,49], an invertebrate laccase-type pathway has also been documented [50]. However, aside from a likely role in cuticle formation, roles of the laccase-type pathway remain to be elucidated [46,50].…”