Identification and function of new proteins in calcified endoskeleton: A new insight in the calcification mechanism of soft corals

Understanding the functional properties of endoskeletal proteins in the calcification processes of soft corals is essential. However, separation of proteins from soft corals is difficult due to contamination by soft tissues and the high sensitivity of soft tissues to handling. We have resolved this problem and established a simple and effective method-electroelution treatment-for purifying proteins from soft corals. Here we applied this newly developed technique to successfully identify four proteins (MPL-1, MPL-2, MPL-3 and MPL-4) from the endoskeletal sclerites of soft coral, Lobophytum crassum as a model. Following this method, we identified a carbonic anhydrase (CA) domain in a soft coral; CA is a key enzyme in living organisms. We found that two CA proteins, which were purified by electroelution, could control the morphology of the CaCO₃ crystals, and one of these is potentially involved in the process of biocalcification. We report here a single protein (MPL-2), which has both calcium-binding and CA activities and is responsible for CaCO₃ nucleation and crystal growth. The sequence of protein MPL-2 was subjected to bioinformatics analysis involving identification of similarities to other animals´ protein. The function of proteins during bio-calcification was also studied by simulating the nucleation and growth of calcium carbonates in vitro. After precipitation of CaCO₃ in the experimental design, the obtained crystals were characterized by X-Ray Diffraction (XRD). These findings suggest that the proteins, which were purified from the calcified sclerites, can control the morphology of CaCO₃ crystals and are potentially involved in the process of bio-calcification.