진주층 생광물화에서 진주조개 Pif 단백질의 기능적 특성 연구

Abstract

Nacre is the inner layer of mollusc shell consisting of a highly organized calcium carbonate composite that provides unique fracture toughness and an iridescent color. Organisms elaborately control the formation of this biomineral using organic macromolecules. Pif, a matrix protein of pearl oyster Pinctada fucata, which post-translationally cleaved into Pif97 (N-terminal) and Pif80 (C-terminal), has been regarded as a key regulator in nacre formation. However, further characterizations have been restricted by the difficulty of isolating individual Pif proteins from nacreous layer. In this thesis, the functional roles of Pif97 and Pif80 in nacre biomineralization were investigated based on experiments employing the recombinant forms which were biosynthesized in Escherichia coli. First, the participation of Pif97 in nacre biomineralization was investigated using recombinant form. We observed that recombinant Pif97 was able to efficiently form a complex with calcium ions. Additionally, recombinant Pif97 showed both in vitro growth inhibition of thermodynamically stable calcite, stabilization of amorphous calcium carbonate, and exclusive binding affinity to metastable aragonite and chitin. These results imply the participation of Pif97 in preventing unwanted crystallization and in the association of the inorganic phase and polysaccharide template. We propose that Pif97 is significant for proper growth of aragonite and for interrelating with organic matrix and inorganic calcium carbonate during nacre biomineralization. Next, the involvement of Pif80 in nacre biomineralization was investigated. Through interactions with calcium ions, recombinant Pif80 participated in the formation of polymer-induced liquid precursor-like amorphous calcium carbonate granules and stabilized these granules by forming calcium ion-induced coacervates. In the calcification condition, the disruption of recombinant Pif80 coacervates destabilized the amorphous mineral precursors, and the re-dissolved recombinant Pif80 controls the growth of aragonite on the polysaccharide substrate, which contributes to the polygonal tablet structure of nacre. We propose that Pif80 plays a role in the development of the inorganic phase during nacre formation process from an ionic state, through an amorphous mineral precursor, to a crystalline biomineral. Collectively, in this dissertation, the biological roles of Pif97 and Pif80 was investigated in nacre biomineralization using molecular biotechnology tools. We propose that each recombinant form has inherent characteristics of the native form, and the novel characteristics of recombinant Pifs provide insight into the use of organic macromolecules by living organisms in nacre biomineralization.