Characterization of a gene family associated with calcified structures in the blue crab, Callinectes sapidus

Eleven cDNAs from a family of genes were cloned from the calcified exoskeleton of the decapod crustacean Callinectes sapidus. Multiple, variant copies of a conserved 18-residue motif (xLxGPSGffxxDGxxxQf), unique to calcified crustacean exoskeleton, accounts for ~70% of the total amino acid residues. The proteins appear to be post-translationally cleaved by a trypsin-like serine protease at conserved recognition sites (RxKR). Two to six peptides, each containing either two or four copies of the 18-residue motif, are expected, depending on which pro-protein is cleaved. Expression of the CsproCP gene family begins at the onset of calcification in the hypodermis of post-ecdysial, calcified cuticle, as shown by Northern analysis. The genes are never expressed in the hypodermis of the noncalcified arthrodial membrane. Western analysis, using an antibody against the 18-residue motif, shows that accumulation of peptides with this motif begins in the calcified cuticle several hours post-ecdysis and continues to anecdysis. The size of the detected peptides agrees with the presumed post-translational cleavage. The strong antibody binding to calcified cuticle proteins and the lack of binding to arthrodial membrane proteins from anecdysial crabs is consistent with immunohistochemical staining performed by Hequembourg (2002). Interestingly, the antibody also weakly binds to proteins from the tendon, another calcified structure in the crab. These results confirm that the proteins encoded by the CsproCP gene family are associated with calcification in Callinectes sapidus.