6533b82efe1ef96bd1293992

RESEARCH PRODUCT

Production, fonction et localisation d'Orchestine: calciprotéine spécifique de la matrice organique des structures minéralisées élaborées par le crustacé terrestre Orchestia cavimana

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description

As most Crustaceans, Orchestia cavimana possesses a mineralized exoskeleton which is periodically replaced. Because of the terrestrial behaviours of this animal, this molting cycle is related to calcium storage and resorption processes. Calcium storage occurs, as calcareous concretions, in diverticula of the midgut called posterior cæca. Calcareous concretions are essentially composed of amorphous calcium carbonate precipitated within a proteinaceous organic matrix composed of a soluble fraction and an insoluble one in an EDTA-buffer. Among the soluble components of the organic matrix, a previous study led to characterize a polypeptide of 23 kDa in SDS-PAGE called Orchestin. This protein, whose gene has been cloned and sequenced, is unglycosylated and binds calcium. The aim of this work was to further characterize this protein marker. The results obtained demonstrated that Orchestin is phophorylated on serine and threonine residues. In order to study the relation between these phosphorylations and the calcium-binding ability of this protein, we produced a recombinant protein devoid of post-translational modification. The comparison of the calcium-binding ability of the native, dephosphorylated (by specific phosphatases) and recombinant proteins led us to show that phosphorylations on the serine residues are of great importance in this ability. Moreover, Orchestin interacts with calcite crystal growth in an in vitro precipitation experiment. On the other hand, the recombinant protein permitted us to explain the discrepancy of molecular masses observed between the native (23 kDa) and sequence deduced (12,4 kDa) proteins. We could thus conclude to the correspondence between the gene orchestin and the protein Orchestin. Finally the recombinant protein was used to produce antibodies in order to locate Orchestin in the biomineralized structures elaborated by O. cavimana during its molting cycle. Orchestin is not only located in the non-mineralized layers of the calcareous concretions (storage structures) but also in the same layers of the calcified spherules (mineralized structures elaborated after ecdysis to resorb the stored calcium). The physical-chemical features exhibited by Orchestin strongly suggest that this calcium-binding phosphoprotein is a key-molecule in the formation of the storage and reabsorption mineralized structures cyclically elaborated by O. cavimana.