KENNEDY, P.J.; SHAFER, T.H.; University of North Carolina at Wilmington: Cloning of a cDNA encoding a putative calcification-associated protein in the cuticle of the blue crab, Callinectes sapidus

The decapod crustacean cuticle is an ideal system for studying biomineralization because growth requires the periodic shedding of the old exoskeleton and calcification of a new one in a well-defined spatial and temporal pattern. During the biomineralization of invertebrate exoskeletons, acidic proteins provide sites for nucleation and control of crystal growth. Andersen and colleagues have isolated 38 insoluble cuticular and arthrodial membrane proteins from Homarus americanus and Cancer pagurus. Over half of the cuticle proteins contain an 18-residue motif (xLxGPSG&phi&phixxDGxxxQ&phi). This motif was found only in calcified cuticle proteins rather than in the proteins of uncalcified arthrodial membrane. Immunohistochemistry of the blue crab, Callinectes sapidus, cuticle demonstrated similar expression patterns. These results suggest that the motif plays an important role in calcification. We cloned a 942 bp full-length cDNA from dorsal hypodermis of the blue crab using 3�- and 5�-RACE and cDNA library screening. This cDNA contains a 708 bp open-reading frame coding for 219 amino acids including a 16-residue signal peptide and 7 variant copies of the 18-residue motif. The inferred translated 23 kDa protein has an isoelectric point of 5.87 and shows strong homology to many of the H. americanus and C. pagurus proteins. Three distinct regions of the open-reading frame of this cDNA are each homologous to different clusters of all the 18-residue-containing C. pagurus proteins. This implies that the previously isolated cuticle proteins are cleaved from a single gene product. This gene�s function in the calcification of the C. sapidus cuticle will be further elucidated by Northern and Western blot analysis throughout the molt-cycle and between cuticle types.