Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus 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. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase-Rabs, and clathrin, are identified by proteomics. A goethite-binding experiment coupled with proteomics and RNA-seq highlights six chitin-binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. 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.

Background Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. Results In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. Conclusions A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.

Biominerals are directed by macromolecules, especially proteins, and incorporate organic occlusions contributing to their overall mechanical properties. However, their distribution patterns in biominerals at the microscale are unclear. Here, we labeled nacre proteins of the pearl oyster Pinctada fucata with cyanine 5 and then directly observed their distribution inside the whole synthetic calcite by using stochastic optical reconstruction microscopy (STORM). Compared to traditional microscopy, we found three forms of occlusions in the crystals, i.e., chain-like, islet-like, and haze-like clusters. In addition, in the whole calcite, a two-ring pattern occurred. Based on the images, we studied the crystal growth process in the presence of nacre proteins with an overgrowth experiment. This study provides a novel approach to directly observe the distribution of nacre proteins in the whole calcite, which has implications for understanding the structures and mechanisms of formation of biominerals as well as for the development of nacre-like materials.

Kinase-family with sequence similarity 20, member C (Fam20C) is a protein kinase, which can phosphorylate biomineralization related proteins in vertebrate animals. However, the function of Fam20C in invertebrate animals especially the role in biomineralization is still unknown. Herein, we cloned the cDNA of fam20C from the pearl oyster, Pinctada fucata. It is showed that the expression of fam20C in the mantle edge was much higher than other tissues. In situ hybridization showed that fam20C was expressed mostly in the outer epithelial cells of the middle fold, indicating it may play important roles in the shell formation. Besides, fam20C expression increased greatly in the D-shape stage of pearl oyster development, when the shell was first formed. During the shell repair process, the expression level of fam20C increased 1.5 times at 6 h after shell notching. Knockdown of fam20C in vivo by RNA interference resulted in abnormally stacking of calcium carbonate crystals at the edges of nacre tablets, showing direct evidence that fam20C participates in the shell formation. This study provides an insight into the role of kinase protein in the shell formation in mollusk and broaden our understanding of biomineralization mechanism.

Phosphorylation of shell matrix proteins is critical for shell formation in vivo and can modulate calcium carbonate formation in vitro.