limpet teeth LC-MSMS - The Microstructure, Proteomics and Crystallization of the Limpet Teeth

Limpets are marine mollusks using mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focuses on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. In this study, we investigated the teeth of limpet Cellana toreuma from three perspectives: 1) by using electron microscopy to observe the microstructure of the teeth; 2) by using proteomics and RNA-seq to investigate the proteins involved in the limpet teeth and 3) by in vitro crystallization experiment combined with Raman spectroscopy to investigate the effects of proteins and chitin framework on crystal formation. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles were densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemical synthesized goethite were loosely packed in the trailing regions of the cusps. Proteins extracted from the whole teeth such as ferritin, peroxiredoxin, arginine kinase, GTPase-Rabs and clathrin were identified by proteomics. Goethite-binding experiment coupled with proteomics and RNA-seq highlighted six chitin-binding proteins (CtCBPs). Furthermore, these proteins or the framework chitin only induced packing of crystals without affecting their crystal polymorphs in vitro. Taken together, the limpets formed hierarchical teeth across different length scales through preformed framework and secreted complex proteins; in addition, the chitin could also be an important player in controlling crystallinity and crystal packing in vivo. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.