CHARACTERIZATION OF THE SKIN MELANOGENETIC SYSTEM OF RANA ESCULENTA L

Tyrosinase is the key enzyme in melanin biosynthesis.We have characterized the skin tyrosinase of Rana esculenta L. in extracts of dorsal skin and by cloning the gene for its functional expression in a heterologous system. Analysis of R. esculenta tyrosinase ORF showed that it is particularly well conserved as compared with other amphibian tyrosinases, but the levels of homology remain high with respect to mammal tyrosinases. The frog enzyme maintains the typical characteristics of tyrosinases, namely the presence of an EGF-like domain, inserted in a laminin–like EGF domain, 15 conserved cysteine residues grouped in 3 clusters, two copper binding sites (CuA and CuB), and a peptide signal for its entry into the RER. Tyrosinase is expressed in HEK293T cells as an active enzyme and its activity is not increased by the action of proteases, and is only slightly increased by copper. Assays of tyrosinase activity on nondenaturing SDS-PAGE demonstrate the presence of two bands, with a molecular weight of approximately 63 kDa and 67 kDa. Conversely, the skin extracts of R. esculenta are lightly active after extraction and need to be activated by proteases like nagarse to reach maximal activation levels. The degree to which these extracts can be activated shows seasonal fluctuations, reaching maximum levels in winter. Assays of tyrosinase activity on SDS-PAGE demonstrate a dopa positive band, with a molecular weight of approximately 200 kDa and a high aggregate that does not migrate in electrophoresis gel in samples treated with nagarse. The different behaviour of the two forms of tyrosinase suggest the formation in vivo of an aggregate containing tyrosinase as shown by its dopa positivity. The proteolytic activation of the skin tyrosinase could probably depend on the exposure of domains favoring the formation of active multimeric complexes, like those already hypothesized in mammals or/and removal of some inhibitor.