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[BPT03-08] Functional Role of Nacrein in Molluscan Shell Biomineralization: Forming CaCO3 Cluster and Regulating Trace Metal Incorporation
Keywords:nacrein, biomineralization, calcium carbonate, protein, vital effect
Pinctada fucata is a marine shellfish utilized for pearl aquaculture and serves as a model organism for biomineralization research. The shell of P. fucata consists of approximately 95% calcium carbonate (CaCO3) and 5% organic matrices, including proteins and sugars, which regulate shell crystallization. Nacrein is the most abundant soluble protein in the shell and contains two functional domains: a carbonic anhydrase (CA) domain, which catalyzes the conversion between CO2 and HCO3−, and G (glycine)-X (mainly acidic amino acids)-N (asparagine) domain, a low-complexity region with structural flexibility. Previous studies have demonstrated that the GXN domain inhibits CaCO3 precipitation in solution; however, the functional role of nacrein in shell formation remains experimentally unclear.
To elucidate the function of nacrein in biomineralization, we measured the optical density of solutions containing Ca2+, CO32−, and nacrein, revealing that nacrein inhibited CaCO3 precipitation in a dose-dependent manner. While other biomineralization proteins exhibit similar inhibitory activity at micromolar concentrations, nacrein functions at nanomolar levels, indicating its strong stabilizing effect on CaCO3 in solution. During the binding of CaCO3, nacrein forms CaCO3 clusters—amorphous aggregates of CaCO3 molecules. The titration experiments of Ca2+ in solutions containing CO32- and nacrein showed that the binding ratio of nacrein to CaCO3 was approximately 1:20,000. Since nacrein has less than 20,000 carboxyl groups, our results suggested that a single nacrein molecule binds to a CaCO3 cluster.
Since the GXN domain’s terminal residues are fixed within the CA domain, we investigated the significance of the dual-domain structure of nacrein. We prepared recombinant proteins with and without fixed terminal residues and used bovine carbonic anhydrase as a model of CA domain. These proteins were mixed with Ca2+ and Mg2+ followed by the incubation under a controlled CO2 atmosphere. Using inductively coupled plasma mass spectrometry (ICP-MS), we found that the CA domain promoted Mg incorporation into CaCO3 crystals, whereas nacrein inhibited Mg incorporation. The GXN domain alone also suppressed Mg incorporation, and the effect was further enhanced when its terminal residues were fixed. This suggested that the CA domain produced inhomogeneous Mg-contaminated CaCO3 by drastically increasing CO32- concentration, whereas the GXN domain facilitates Ca2+ aggregation, forming pure CaCO3 clusters. X-ray diffraction (XRD) analysis confirmed that CaCO3 precipitates in the presence of nacrein became pure crystal with low density of crystal defects.
In conclusion, nacrein plays a crucial role in biomineralization by providing pure CaCO3 clusters at calcification sites, thereby ensuring precise control over trace element incorporation. This study suggested that organic molecules involved in biomineralization may contribute to the regulation of vital effects in living organisms.
To elucidate the function of nacrein in biomineralization, we measured the optical density of solutions containing Ca2+, CO32−, and nacrein, revealing that nacrein inhibited CaCO3 precipitation in a dose-dependent manner. While other biomineralization proteins exhibit similar inhibitory activity at micromolar concentrations, nacrein functions at nanomolar levels, indicating its strong stabilizing effect on CaCO3 in solution. During the binding of CaCO3, nacrein forms CaCO3 clusters—amorphous aggregates of CaCO3 molecules. The titration experiments of Ca2+ in solutions containing CO32- and nacrein showed that the binding ratio of nacrein to CaCO3 was approximately 1:20,000. Since nacrein has less than 20,000 carboxyl groups, our results suggested that a single nacrein molecule binds to a CaCO3 cluster.
Since the GXN domain’s terminal residues are fixed within the CA domain, we investigated the significance of the dual-domain structure of nacrein. We prepared recombinant proteins with and without fixed terminal residues and used bovine carbonic anhydrase as a model of CA domain. These proteins were mixed with Ca2+ and Mg2+ followed by the incubation under a controlled CO2 atmosphere. Using inductively coupled plasma mass spectrometry (ICP-MS), we found that the CA domain promoted Mg incorporation into CaCO3 crystals, whereas nacrein inhibited Mg incorporation. The GXN domain alone also suppressed Mg incorporation, and the effect was further enhanced when its terminal residues were fixed. This suggested that the CA domain produced inhomogeneous Mg-contaminated CaCO3 by drastically increasing CO32- concentration, whereas the GXN domain facilitates Ca2+ aggregation, forming pure CaCO3 clusters. X-ray diffraction (XRD) analysis confirmed that CaCO3 precipitates in the presence of nacrein became pure crystal with low density of crystal defects.
In conclusion, nacrein plays a crucial role in biomineralization by providing pure CaCO3 clusters at calcification sites, thereby ensuring precise control over trace element incorporation. This study suggested that organic molecules involved in biomineralization may contribute to the regulation of vital effects in living organisms.