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Abstract:
Protein phosphorylation is a fundamental mechanism regulating many aspects of cellular processes. Shell matrix proteins (SMPs) control crystal nucleation, polymorphism, morphology, and organization of calcium carbonate crystallites during shell formation. SMPs phosphorylation is suggested to be important in shell formation but the mechanism is largely unknown. Here, to investigate the mechanism of phosphorylation of SMPs in biomineralization, we performed in vivo and in vitro experiment. By injection of antibody against the anti-phosphoserine/threonine /tyrosine into the extrapallial fluid of the pearl oyster Pinctada fucata, phosphorylation of matrix proteins were significantly reduced after 6 days. Newly formed prismatic layers and nacre tablet were found to grow abnormally with reduced crystallinity and possibly changed crystal orientation shown by Raman spectroscopy. In addition, regeneration of shells is also inhibited in vivo. Then, protein phosphatase was used to dephosphorylate SMPs extracted from the shells. After dephosphorylation, the ability of SMPs to inhibiting calcium carbonate formation have been reduced. Surprisingly, the ability of SMPs to modulate crystal morphology have been largely compromised although phosphorylation extent remained to be at least half of the control. Furthermore, dephosphorylation of SMPs changed the distribution of protein occlusions and decreased the amount of protein occlusions inside crystals shown by confocal imaging, indicating interaction between phosphorylated SMPs and crystals. Taken together, this study provides insight into the mechanism of phosphorylation of SMPs during shell formation.
