Quantitative proteomics provides new insights into chicken eggshell matrix protein functions during the primary events of mineralisation and the active calcification phase

Remerciements : GenoToul, Plateforme Bioinformatique de Toulouse Midi-Pyrénées; International audience; Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units 4) and development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised. We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation. Significance The avian eggshell is a natural protective barrier of the egg as long as it remains intact. It prevents microbial penetration to ensure a harmonious development of the embryo and table eggs free of pathogens for consumers. The eggshell is a calcitic biomineral structure initiated on organic nucleation sites which are evenly disposed on eggshell membranes. Its structural organisation results from an interaction between matrix proteins and calcium carbonate, in particular calcite crystal during the growth phase. The initial steps of the calcification process during which calcitic mineral phase is selected and the process of crystal orientation selection by competition for space are crucial in the determination of the whole eggshell columnar ultrastructure and its resulting exceptional mechanical properties. Recent studies have also described the physical characteristics of calcium carbonate during the initial sequence of calcium carbonate biomineralisation in many species and have highlighted the key role of amorphous calcium carbonate. In order to determine precisely the importance of particular proteins relative to these key events at the onset of mineralisation, this project used quantitative proteomics of matrix proteins in forming eggshell, assuming that a protein more abundant at a particular stage of the process has an higher probability to be involved in its control. This information was coupled with bioinformatic analysis predicting the function of proteins in the stabilisation of disordered forms of calcium carbonate and their influence on crystal polymorph and morphology. Eggshell calcification is a relevant model for studying calcium carbonate biomineralisation on a membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressures and temperatures and might provide means of reinforcing its solidity.