Purification, Characterization, and in VitroMineralization Studies of a Novel Goose Eggshell Matrix Protein, Ansocalcin

Abstract

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 g/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentrationdependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.Organisms are capable of developing minerals and biocomposites with complex architecture to fulfill important biological functions, such as skeletal support, protection of soft tissues, and food grinding (1-4). Often, survival of the organism depends on the structure and strength of these composite materials. Due to the wide range of mechanical and functional properties of these biocomposites, identifying the organic and inorganic components and understanding the structure-function relationships have potential industrial and biotechnological applications (5). The inorganic mineral phase of such materials is formed over an insoluble organic matrix or mold, and the mineral phase is intimately associated with organic macromolecules, such as proteins/glycoproteins, polysaccharides, or proteoglycans (6). These biomacromolecules are highly acidic in nature and have been postulated to control nucleation, growth, crystal size, and shape of the mineral phases (7).Mann (8) classified the biologically programmed composites into four types (types I, II, III, and IV), based on matrix intervention in nucleation and growth processes. Avian eggshells form type II biocomposites, which is the fastest forming hard acellular composite in nature. For example, in the case of chicken eggshell, about 5 g of the mineral phase is produced within 24 h (9). Their calcified layer consists of ϳ95% mineral and ϳ5% organic phase (10). The ...

In vitro effects of recombinant otoconin 90 upon calcite crystal growth. Significance of tertiary structure

“…Fetuin A has long been known to play an important role in inhibition of ectopic apatite formation (Schafer et al, 2003). Also the eggshell protein ansocalcin leads to increased nucleation, but in addition, causes marked crystal aggregation (Lakshminarayanan et al, 2003). …”

Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata

“…Information on the structure-function relationship of matrix proteins and their role in calcification may add a piece to the puzzle of how the matrix shell, a superior mechanical material, grows (56). However, relatively few shell matrix proteins have been identified and characterized and their roles in mineralization evaluated.…”

Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens

“…The OC-17 and related proteins identified in the goose (ansocalcin) (Lakshminarayanan et al, 2003), ostrich (struthiocalcin) (Mann and Siedler, 2004) or in molluscs (perlucin) (Weiss et al, 2000) are lectin-like proteins which may act as framework proteins but their involvement in mechanical properties has not yet been established. The improvement in shell breaking strength after moult was positively correlated with the change in concentration of OC-17 (Table 4) suggesting a possible involvement of this protein.…”