Identification of Matrix Proteins Affecting Crystal Texture in chicken and guinea fowl eggshells – IMPACT

The chicken eggshell is a complex mixture of proteins which interact with calcium carbonate to allow the formation of a precise and ordered crystalline structure which give to the egg its mechanical properties. Amongst more than 600 proteins potentially involved, we have performed quantitative approaches to characterise and determine molecules involved at various key stages of shell mineralisation.
Methods used quantitative proteomics and RNA sequencing, and associated to physical measurements to characterise eggshell mineralogy.
Theses multidisciplinary and complementary approaches revealed the matrix proteins which are responsible for shell texture, and yield insight into protein functionality in eggshell mechanical properties to provide natural defence of the egg against bacterial penetration and to resist physical environmental insults.

We have demonstrated for the first time in vertebrates, the presence of amorphous calcium carbonate (ACC) as a transient mineral phase, more soluble and reactive. This amorphous phase allows to explain the rapid calcification process of the chicken eggshell.
Two qualitative and quantitative studies were also performed and determined more than 300 shell matrix proteins involved in these calcification events. Amongst then, we have sorted about 20 important candidates for which their functions would be important in the process of shell formation and the amorphous mineral transformation in crystalline counterpart.

Further experiments will be performed to analyse molecular genetic sequences (RNA) coding for these proteins. Furthermore, the same approach will be developed for Guinea fowls which product exceptionally strong eggshells. These approaches will allow us to establish common and differences molecular mechanisms between both species, and will give new insight to better understand biomineralisation mechanisms

During the last 18 months, 5 articles were published in international peer reviewed journals. Furthermore this project and the corresponding results were presented in 2 international and various national congress and conferences.

The calcitic avian eggshell protects the developing embryo and ensures that the nutritious table egg remains free of pathogens. Eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes), followed by an interaction between the growing mineral crystals and the shell organic matrix, which lead to a highly ordered microstructure and texture of the resulting shell with exceptional mechanical properties. Shell mineralisation occurs in 3 distinct phases (initiation, growth and completion), which are associated with distinct matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The eggshell matrix is a complex mixture of proteins, proteoglycans and glycoproteins, which control the nucleation and crystal growth phases of mineralisation to determine the textural and mechanical properties of the shell. The recent development of high-throughput methods has led to the identification of sequences associated with eggshell mineralisation (528 proteins and 605 transcripts), but little is known concerning their function. In order to determine precisely their importance relative to eggshell mineralization, this project will use quantitative proteomics and RNA sequencing methodologies, with bioinformatic analysis and correlation to eggshell mineralogical composition and textural characteristics, to elucidate the pivotal role of eggshell matrix proteins in the establishment of crystal nucleation, shell microstructure, texture and mechanical properties.
Task 1 will use extracts from chicken eggshell and tissues involved in calcification, collected at various stages of shell formation, to define proteins and transcripts associated with each phase of calcification, and more particularly with the initiation of mineralization. Statistical analysis will establish the restricted proteins and transcripts that are strongly correlated with the initiation phase and consequently are highly likely to be responsible for the first crystal nucleation events (primary nucleation). In task 2, a comparative analysis of both chicken and guinea fowl matrix proteins will be performed. The guinea fowl shell is exceptionally strong due to altered crystal texture, which arises from secondary nucleation in the middle of the shell layer due to specific, as yet uncharacterized, matrix components. A uterine cDNA library will be prepared and analysed to obtain sequences of thousands of guinea fowl gene products. This approach will be complemented by exhaustive proteomics (possible with the cDNA sequence information) and additional de novo sequencing. The quantitative proteomic and RNA sequencing performed on samples collected at different phases of shell formation will allow determination of the proteins responsible for the key shift in crystal orientation, specifically associated with guinea fowl eggshell texture and the resulting enhanced mechanical properties. In task 3, the interaction between calcium carbonate and proteins collected during the initiation phase and additional stages of shell formation will be determined in chicken and guinea fowl samples using an in vitro crystal growth assay. The in situ texture and mechanical properties of the eggshells collected at the different stages will be measured. In silico bioinformatics will be performed (Task 4) to determine sequence motifs and domains associated with mineralisation. Integration of this data will elucidate the mechanisms controlling the mineralogy and microstructure of the forming shell.
This multidisciplinary approach will characterize the essential matrix components responsible for the different phases of shell formation, and will provide fundamental information concerning proteins that control primary and secondary nucleation events during calcitic biomineralisation. These results will yield new insights into how proteins regulate the mechanical properties of the eggshell to provide the primary natural defence of the egg against microbial penetration.