Phosphorylation of serine residues is fundamental for the calcium-binding ability of Orchestin, a soluble matrix protein from crustacean calcium storage structures.

Orchestia cavimana is a terrestrial crustacean, which cyclically stores calcium in diverticula of the midgut, in the form of calcified amorphous concretions. These concretions are associated with a proteinaceous matrix, the main constituent of the soluble matrix is Orchestin, an acidic calcium-binding protein [Testenière et al., Biochem. J. 361 (2002) 327-335]. In the present paper, we clearly demonstrate that Orchestin is phosphorylated on serine and tyrosine residues, but that calcium binding only occurs via the phosphoserine residues. To our knowledge, this is the first example of an invertebrate mineralization for which a post-translational modification is clearly related to an important function of a calcifying protein.

Orchestin, a calcium-binding phosphoprotein, is a matrix component of two successive transitory calcified biomineralizations cyclically elaborated by a terrestrial crustacean.

Orchestia cavimana is a crustacean that cyclically replaces its calcified cuticle during molting cycles in order to grow. Its terrestrial way of life requires storage of calcium during each premolt period, as calcareous concretions, in tubular diverticula of the midgut. During the postmolt period the stored calcium is reabsorbed and is translocated through the storage organ epithelium as calcified small spherules. In a previous study, we sequenced and characterized a remarkable component of the organic matrix of the premolt storage structures, Orchestin, which is a calcium-binding phosphoprotein. In this paper, we analyzed the spatiotemporal expression of the orchestin gene by Northern blotting and in situ hybridization, and its translated product by immunocytochemistry. We found evidence that the gene and the protein are expressed specifically during premolt in the storage organs. More interestingly, we demonstrated that the protein is synthesized also during the postmolt period, as a component of the organic matrix of the calcium resorption spherules. Thus, Orchestin is a matrix component that is synthesized by the same cells to contribute alternately to the elaboration of two different calcifications. These results, in addition to the physical and chemical features of the protein, suggest that Orchestin is probably a key molecule in the calcium carbonate precipitation process leading to the cyclic elaboration of two transitory calcified mineralizations by the crustacean Orchestia.

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

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.