| COMM domain-containing or COMMD proteins are a group of recently discovered proteins which are characterized by the presence of a unique copper metabolism gene MURR1 domain (COMMD) of 70 to 85 amino acids in their extreme carboxy termini. This family consists of 10 subgroups ,COMMD1-COMMD10. With the exception of COMMD1, little information is now available regarding the biochemical characteristics and functions of the other members, especially in lower organisms. Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. It is well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis. Sequencing of the whole genome of Florida amphioxus offers a new opportunity to the research of molecular and evolutionary biology of amphioxus. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of BbCOMMD6 from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtaunese.The cDNA encoding B. belcheri COMMD6, BbCOMMD6, obtained from the cDNA library of Branchiostoma belcheri is 1073 bp long. Its longest ORF is 612 bp encoding for a 203 amino acids long protein with a predicted molecular mass of approximately 23 kDa. BbCOMMD6 has a COMM domain at the residues 116-201 at its C-terminal region and an extended N-terminal portion. Alignment analysis showed that the COMM domain of BbCOMMD6 shared 47.0%, 45.3%, 44.7%, 44.2%, 45.8%, 43.5%, 54.7%, 50.0% and 44.4% identity to that of human, chimpanzee, house mouse, dog, cattle, frog, zebrafish, sea urchin and slime mold COMMD6 proteins, respectively. However, its N-terminal region consisted of 115 amino acids, was close to that of frog, zebrafish and sea urchin COMMD6 proteins in length, and exhibited 28.2%, 27.9% and 40.9% identity to frog, zebrafish and sea urchin COMMD6 proteins in sequence, individually, contrasting to the significantly shorter N-terminal portion of mammalian COMMD6 proteins. The phylogenetic tree constructed using the sequences of representative COMM domain-containing proteins including BbCOMMD6 demonstrated that BbCOMMD6 was clustered with COMMD6 proteins, separating from all the other 9 family members.An expression vector including the entire ORF of BbCOMMD6 and a 5'additional tag of pET30a is constructed and transformed into E.coli. Recombinant protein is expressed and purified. The purified protein with His6 tag yielded a single band of ~28 kDa under SDS-PAGE in the presence ofβ-mercaptoethanol, but occurred as two bands in the absence ofβ-mercaptoethanol, with one band being a molecular mass of ~28 kDa and the other being ~55 kDa. These indicated that the purified BbCOMMD6 existed as a mixture of monomers and dimers generated via disulfide bond formation. After the preincubation with CuCl2, BbCOMMD6 was analyzed by native-PAGE and SDS-PAGE withoutβ-mercaptoethanol. The results showed that the presence of Cu(II) can't enhance recombinant BbCOMMD6 dimer formation. Western blot analysis of the cytosolic and mitochondrial fractions from B. belcheri with rabbit anti-BbCOMMD6 serum showed that BbCOMMD6 was present in the cytosolic fraction. This indicated that BbCOMMD6 was mainly localized in the cytosol. Both the pull-down and reverse pull-down assays revealed that recombinant BbCOMMD6 can interact with the creatine kinase (CK), an essential enzyme involved in energy metabolism, forming a heterodimer BbCOMMD6-CK; and the enzymatic activity assays showed that both MM-type and BB-type CK activities are inhibited by BbCOMMD6 in a dose-dependent manner. All these data suggest that BbCOMMD6 may be involved in energy transduction, via binding to CK and inhibiting activities of CK, and offer first clues to its role as a regulator of CK activities. |
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