Purification and functional analysis of the shell matrix protein N66 from the shell of the pearl oyster Pteria sterna

Mollusk biomineralization is a process controlled by a complex interplay of proteins, ions and external regulators. In spite of several studies, there is a lack of knowledge of who (molecules involved), how (mechanism) and why (evolution and adaptation) mollusk are designed as we know them. In this study, a shell matrix protein, N66, has been purified and characterized biochemically from the shell of Pteria sterna. Two protein bands with carbohydrates associated were separated with a molecular weight of similar to 60 and 64 kDa. It has carbonic anhydrase activity and it is able to form crystal polymorphs of calcium carbonate in vitro. The mRNA N66 was obtained from the mantle tissue of Pteria sterna and the deduced amino acid sequence contained a carbonic anhydrase (CA) domain and a Asn/Gly-rich domain (aa243-439). The CA domain contained three His residues acting as zinc ligands and the gate-keeper residues present in all alpha-CAs (Glu(166)-Thr(525)), being thus similar to the human isoform hCAVII. Also, to test whether the posttranslational modifications present on the native N66 affects the CA activity and its crystallization capability in vitro, a recombinant N66 was overexpressed in Escherichia coli and functionally characterized. Our results show that recombinant N66 has higher CA activity and produce larger size crystals in vitro than the native N66 protein, suggesting that intrinsic properties of the native N66, such as glycosylations and/or phosphorylations, might regulate its activity.