Characterization and spatiotemporal expression of orchestin, a gene encoding an ecdysone-inducible protein from a crustacean organic matrix.

Abstract

We report the characterization of a new gene encoding an acidic protein named Orchestin. This protein is a component of the organic matrix of calcium storage structures (calcareous concretions) elaborated during the moulting cycles of the terrestrial crustacean Orchestia cavimana. The deduced molecular mass of Orchestin is estimated to be 12.4 kDa and the pI to be 4.4, whereas the native protein extracted from the calcium deposits migrates as a 23 kDa band on SDS/PAGE. This discrepancy is probably due to the richness of this protein in acidic amino acids (approx. 30%). The protein obtained by expressing the Orchestin cDNA in Escherichia coli presents an electrophoretic mobility of 25 kDa. Antibodies raised against the recombinant protein recognize the 23 kDa native protein exclusively among the organic-matrix components. Spatiotemporal analysis of the expression of the orchestin gene shows that it is expressed only in the storage organ cells when the concretions are elaborated during the premoult period and also, to a smaller extent, during the postmoult period. The translation products are expressed in accordance with the transcript expression during both the premoult and postmoult periods. Study of the hormonal stimulation of orchestin reveals that 20-hydroxyecdysone induces this gene as a secondary-response or late-response gene.