Cloning, Expression And Functional Analysis Of Creatine Kinase Gene In Amphioxus Branchiostoma Belcheri Tsingtauense

Creatine kinase (CK) belongs to the family of ATP:guanidine phospho- transferases whose members play important roles in intracellular energy flow. CK is widely distributed in organisms ranging from sponge to vertebrates. Little is known to date about CK in the amphioxus Branchiostoma belcheri. Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. In this paper, we report expression and functional analysis of CK gene in amphioxus B. belcheri tsingtauense.The cDNA encoding B. belcheri CK, BbCK, obtained from the cDNA library of Branchiostoma belcheri is 1787 bp long. Its longest ORF was 1140 bp encoding for a 379 amino acids long protein with a predicted molecular mass of approximately 43 KDa. BbCK has no N-terminal signal peptide. Like vertebrate CKs, BbCK contains ATP:guanido phosphotransferase N-terminal domain and ATP: guanido phosphotransferase C-terminal catalytic domain, and most of the catalytic residues are found in the larger C-terminal domain. ATP: guanido phosphotransferases active site contains contains consensus sequence CPSNLGT. Alignment of BbCK with CKs of other species revealed that the CK family is well conserved, and BbCK is most similar to Mi-CK of other species.Insitu hybridization histochemistry demonstrated that BbCK transcript was widely distributed in adult amphioxus and most abundant in the hepatic caecum and hind-gut.Quantative real-time PCR showed that challenge with LPS resulted in a quick and significant up-regulation of BbCK expression in adult amphioxus, suggesting that BbCK may be a key acute phase protein in amphioxus and involve in the host immune defense. The mode of action by which BbCK is involved in the immunity is currently underway.