Novel identification of matrix proteins involved in calcitic biomineralization.

Calcitic biomineralization is essential for otoconia formation in vertebrates. This process is characterized by protein-crystal interactions that modulate crystal growth on an extracellular matrix. An excellent model for the study of calcitic biomineralization is the avian eggshell, the fastest known biomineralization process. The objective of this study is to identify and characterize matrix proteins associated with the eggshell mammillary cones, which are hypothesized to regulate the earliest stage of eggshell calcification. Mammillary cones were isolated from 2 models, fertilized and unfertilized, and the released proteins were identified by RP-nanoLC and ES-MS/MS proteomics. Proteomics analysis identified 49 proteins associated with the eggshell membrane fibers and, importantly, 18 mammillary cone-specific proteins with an additional 18 proteins identified as enriched in the mammillary cones. Among the most promising candidates for modulating protein-crystal interactions were extracellular matrix proteins, including ABI family member 3 (NESH) binding protein (ABI3BP), tiarin-like, hyaluronan and proteoglycan link protein 3 (HAPLN3), collagen alpha-1(X), collagen alpha-1(II) and fibronectin, in addition to the calcium binding proteins calumenin, EGF-like repeats and discoidin 1-like domains 3 (EDIL3), nucleobindin-2 and SPARC. In conclusion, we identified several cone-resident proteins that are candidates to regulate initiation of eggshell calcification. Further study of these proteins will determine their roles in modulating calcitic biomineralization and lead to insight into the process of otoconia formation/regeneration. BIOLOGICAL SIGNIFICANCE: Biomineralization is essential for the development of hard tissues in vertebrates, which includes both calcium phosphate and calcium carbonate structures. Calcitic mineralization by calcium carbonate is an important process in the formation of otoconia, which are gravity receptor organs located in the inner ear and are responsible for balance and for sensing linear acceleration. Deficiencies in the regulation of their biomineralization can lead to otoconia degeneration and eventually benign paroxysmal position vertigo (BPPV), which is the main cause of vertigo in humans. Eggshell formation in chicken is one of the fastest known biomineralization processes and is an excellent model for the study of calcitic biomineralization. Cross-analysis of proteomic data from two mineralized models, fertilized and unfertilized chicken eggshells, identified proteins associated with the mammillary cones that are the sites of initiation of eggshell formation. We hypothesize that these proteins regulate the earliest stages of eggshell calcification. The human homologs of these proteins are therefore potential candidates to play a role in otoconia biomineralization.