Research On Biological Function Of Calmodulin-like Protein Cloned From Pearl Oyster Pinctada Fucata

As the product of calcium metabolism, the formation mechanism of pearl and shell of marine bivalve is always the hot field of biomineralization. This research is based on our previous successful cloning of a completely novel calcium metabolism related protein—calmodulin-like protein(CaLP) from pearl oyster (Pinctada fucata) and calmodulin(CaM). The main purpose of this research is to find out the function of CaLP on the cell level and compare functions of these two proteins.During a special biomineralization process—shell regeneration, we found that both CaLP exhibit special functions in shell related secretory vesicles; during the differentiation of osteoblast MC3T3-E1, CaLP exhibite significant effect on its differentiation and biomineralization. Besides, the C-terminal tail of CaLP is very important in this process.By using the method of Western blot (WB) on fractions of nuclear and cytosol of primary cultured mantle cells and immunogold electron microscopy, we found out the completely different localization of these two proteins: CaM was localized both in cytoplasm and nuclear but CaLP was localized only in the cytoplasm. By constructing different mutants and using method of fluorescence confocal microscopy we confirmed that the C-terminal globular domain together with the extra tail of calmodulin-like protein was very important in its sequestration in the cytoplasm.To further investigate functions of different domains of CaLP in the protein-protein interaction, we pick up p21 as the object of protein interaction. By co-immunoprecipitation of CaLP and its different mutant with p21, we confirmed that the C-terminal globular domain of this protein is responsible for its interaction with p21. Another interesting phenomenon is that when CaLP and p21 are co-transfected, the expression of p21 can stimulate CaLP translocate from cytoplasm to nucleus. By using methods of bioinformatics, point mutation and flow cytometry, we confirmed the importance of Y139 in the nuclear-cytoplasmic trafficking of CaLP. CaLP's phosphorylation state analysis of different localization showed that when CaLP is overexpressed alone, it is in state of phosphorylation and stays in the cytoplasm; when CaLP is co-transfected with p21, p21 overexpression causes its unphosphorylation and translocated from cytoplasm to nucleus.