It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls.Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO-formation, thus participating in calcium carbonate crystal formation of the nacreous layer.Crystallization, observed in many organisms, is a genetically regulated process. In the mammal, several factors regulating bone morphogenesis are identified. Osteopontin, an acidic calcium binding protein, is isolated from the mineralized phase of bone (1). Another bone matrix protein, osteocalcin (2), contains 'y-carboxyglutamic acid residues, which participate in calcium binding. In general, the mollusk shell is mainly composed of two layers, a prismatic layer and nacreous layer. Both layers are in the forms of calcium carbonate crystal; however, the prismatic layer forms calcite and the nacreous layer forms aragonite. Such crystal polymorphism in the two closely situated layers is a highly elaborated phenomenon in the mollusk. In such regulated processes, organic matrices secreted from the mantle epithelia have been suggested to play critical roles (3); the major components of soluble organic matrices are aspartic acid-rich calcium binding proteins (4-8). Recently, Falini et al. (9) and Belcher et al. (10) reported that macromolecules extracted from the nacreous shell layers induced aragonite formation in vitro.Pearl oysters (Pinctada fucata) produce pearls inside the shells. Such a pearl is equivalent to the mollusk shell layer, which is composed of the aragonite crystal and organic matrices. Therefore, to isolate the organic matrix protein, we have chosen the pearl as a starting material. Staining with Coomassie brilliant blue and Stains-all (Nacalai Tesque, Kyoto), we have identified a 60-kDa protein that we named nacrein. Analysis of cDNA encoding nacrein revealed that it had a similar domain to carbonic anhydrase (CA). Furthermore, the native nacrein purified from the pearl possessed CA enzymatic activity. To our knowledge, t...