A basic protein, N25, from a mollusk modifies calcium carbonate morphology and shell biomineralization.

Biomineralization is a widespread biological process in the formation of shells, teeth, or bones. Matrix proteins in biominerals have been widely investigated for their roles in directing biomineralization processes such as crystal morphologies, polymorphs, and orientations. Here, we characterized a basic matrix protein, named mantle protein N25 (N25), identified previously in the Akoya pearl oyster (Pinctada fucata). Unlike some known acidic matrix proteins containing Asp or Glu as possible Ca2+-binding residues, we found that N25 is rich in Pro (12.4%), Ser (12.8%), and Lys (8.8%), suggesting it may perform a different function. We used the recombinant protein purified by refolding from inclusion bodies in a Ca(HCO3)2 supersaturation system and found that it specifically affects calcite morphologies. An X-ray powder diffraction (XRD) assay revealed that N25 could help delay the transformation of vaterites (a metastable calcium carbonate polymorph) to calcite. We also used fluorescence super-resolution imaging to map the distribution of N25 in CaCO3 crystals and transfected a recombinant N25-EGFP vector into HEK-293T cells to mimic the native process in which N25 is secreted by mantle epithelial cells and integrated into mineral structures. Our observations suggest N25 specifically affects crystal morphologies and provide evidence that basic proteins lacking acidic groups can also direct biomineralization. We propose that the attachment of N25 to specific sites on CaCO3 crystals may inhibit some crystal polymorphs or morphological transformation.