Investigation of the role of ansocalcin in the biomineralization in goose eggshell matrix

Abstract

The role of proteins in biomineralization and the mechanism of eggshell formation are not well understood. We have isolated and purified the major protein, ansocalcin from goose eggshell matrix. The amino acid sequence study indicates that ansocalcin is homologous to the chicken eggshell protein, ovocleidin 17, and C-type lectins. Ansocalcin nucleates polycrystalline aggregates of calcite crystals in in vitro mineralization experiments. The polycrystalline aggregates obtained at higher concentration of ansocalcin appears to be similar to the crystals observed at the mamillary layer of the eggshell.

Figure 1

Schematic representation of the structure of the eggshell (B) with SEM micrographs showing the cross section (A), and the mamillary caps (see C Inset for detailed picture).

Figure 2

Amino acid sequence homology of ansocalcin with other related proteins. Ovocleidin isolated from chicken eggshell (35), c-type lectin found from snake venom (36). The identical amino acid residues are shaded in black; asterisks indicate the pair of acid residues observed in ansocalcin.

Figure 3

Morphological changes in the calcite crystals grown in the presence of ansocalcin at various levels. (A) Control; (B) 10 μg/ml; (C) 100 μg/ml; (D) 500 μg/ml. See Inset for an enlarged crystal. (Scale bar, 50 μm.)

Figure 4

Size exclusion chromatogram (SEC) of ansocalcin in CaCl₂ solution at various concentrations, 0.1 mg/ml (1) and 0.5 mg/ml (2). The dotted lines represent the position of the standards: (a) BSA (66 kDa), (b) egg white albumin (44 kDa), and (c) chicken egg lysozyme (14 kDa).