Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Abstract

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with L-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. POlike activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4- hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40 kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10 kDa and/or ~200 kDa protein bands. The ~10 kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10 kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves