Molecular characterization of two CuZn-SOD family proteins in the Pacific oyster Crassostrea gigas

Superoxide dismutases (SOD) are multifamily antioxidant enzymes, playing an important role in the defense against oxidative stress in all organisms. Genomic information indicated the presence of genetic diversification of the copper and zinc SOD (CuZn-SOD) family in oysters. In the present research, we characterized two CuZn-SOD family proteins, Cg-CuZn-SOD and Cg-dominin3, in the Pacific oyster Crassostrea gigas using comprehensive sequence analyses, recombinant proteins and site-directed mutagenesis, and observations of gene expression in larval and adult oysters. We found that Cg-CuZn-SOD possessed sequence and structural elements conserved in a CuZn-SOD molecule and the recombinant protein was confirmed empirically to have the SOD enzyme activity. In contrast, Cg-dominin3 lacked five of the seven residues essential for the conformation of SOD active center and the recombinant protein did not have the enzyme activity. However, recombinant Cg-dominin3 showed strong binding activities toward zinc and copper ions. Substitutions of five conserved His residues in the active center demolished the SOD activity but enhanced the metal binding capacity in Cg-CuZn-SOD. On the other hand, reinstallation of the five His residues that were assumed to be activity essential and lost in evolution did not restore the SOD enzyme activity in Cg-dominin3. Additionally, the coding genes of the two proteins exhibited different patterns of expression during larval development and in adult oyster in response to zinc challenges. These results have led to the discovery of the first cytoplasmic CuZn-SOD molecule and the confirmation of molecular diversification of extracellular CuZn-SOD homologs in oysters.

Automated summary

In this study, two copper and zinc SOD family proteins were characterized in the Pacific oyster Crassostrea gigas. The results revealed the presence of a cytoplasmic CuZn-SOD molecule and molecular diversification of extracellular CuZn-SOD homologs in oysters.