A transporter that allows phosphate ions to control the polymorph of exoskeletal calcium carbonate biomineralization

Abstract

The SLC20A2 transporter supplies phosphate ions (P_i) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of P_i are found in the CaCO₃-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and P_i transport to exoskeletal elements was studied with respect to the role of P_i in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for P_i transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by P_i. RNA interference experiments via CqSLC20A2 dsRNA injections reduced P_i content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO₃) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced P_i content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which P_i affects CaCO₃ biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of P_i as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO₃, with P_i in minute amounts • P_i was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for P_i was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced P_i.

Keywords: Amorphous calcium carbonate (ACC); Biomineralization; Crustacean exoskeleton; Phosphate; SLC20 transporter.