Ovocalyxin-32, a Novel Chicken Eggshell Matrix Protein

Abstract

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.

Cloning of Ovocalyxin-36, a Novel Chicken Eggshell Protein Related to Lipopolysaccharide-binding Proteins, Bactericidal Permeability-increasing Proteins, and Plunc Family Proteins

“…Three other eggshell-specific proteins have been localized in the mineralized region of the eggshell. Ovocalyxin-32 is most abundant in the outer shell (16), whereas Ovocleidin-116 is most intensively detected in the palisade layer (13). On the other hand, Ovocleidin-17 is abundantly distributed throughout the mammillary cones and the palisade layer (10).…”

“…SDS-PAGE and electro-blotting were performed on a 4 -20% gradient gel or 12% isocratic gels as already described (2,16 Tween 20), the membranes were incubated for 1 h with 1:5,000 Alexa Fluor 680 goat anti-rabbit IgG (Molecular Probes) in Odyssey blocking buffer. The membranes were then washed four times in PBS/Tween and then twice in PBS.…”

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

“…A number of such biomacromolecules have been extracted, purified, and identified from the chicken eggshells (31)(32)(33)(34)(35)(36)(37)(38). These biomacromolecules, especially the matrix proteins, are believed to be active in controlling the morphology and selective nucleation of polymorphs of the CaCO 3 layer of the eggshell (39).…”

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

“…The organic phase (proteins and proteoglycans) of the eggshell matrix is believed to be responsible for the nucleation and directed growth of the calcified layer. So far, several matrix proteins from chicken eggshells have been purified and characterized (11)(12)(13)(14)(15)(16)(17). These proteins are subdivided into three groups, namely non-collagenous bone proteins (osteopontin), eggshell-specific proteins (ovocleidins and ovocallyxins), and egg white proteins (ovalbumin, ovotransferrin, and lysozyme).…”