Distribution and function of the nacrein-related proteins inferred from structural analysis.

Nacrein is the first identified molluscan organic matrix (OM) component considered to be specifically involved in nacreous layer formation (Miyamoto et al. in Proc Natl Acad Sci USA 93:9657-9660, 1996); however, its localization in shell microstructures and phylogeny of molluscs and function still remain unclear. Therefore, to elucidate the functions of the nacrein-related proteins, we set up three experiments focused on (1) the primary structure of the nacrein-related proteins, (2) the tertiary structure of nacrein, and (3) in vitro crystallization of the proteins. In regard to the first experiment, our Western blot analysis and cDNA cloning clearly indicated for the first time the common occurrence of nacrein-related proteins both biochemically and genetically, independent of molluscan phylogeny and shell microstructures. Together with the data reported so far, we classified nacrein-related proteins into four types. Second, we determined the overall structure of nacrein via small-angle x-ray scattering via the program DAMMIN. This kind of research has never yet been attempted for the molluscan OM proteins. Our results inferred the structure of nacrein to be N-shaped based on the low-resolution solution dummy atom model structures that could be derived from the presence of the NG-repeat domain that was intercalated into two CA domains. Third, the result of the crystallization experiment revealed inhibitory activity of crystal formation for nacrein-related proteins when present in free state but the same molecule, when attached to the ISM, may regulate the form and size of aragonite crystal. These results demonstrate the fundamentally important function of nacrein-related proteins in molluscan shell formation.