Ovocalyxin-32, a novel chicken eggshell matrix protein - Isolation, amino acid sequencing, cloning, and immunocytochemical localization

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell m embranes. Mineralization takes place in an acellular uterine fluid, which c ontains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels i n the uterine and isthmus regions of the oviduct, and concentrated in the e ggshell. Sequencing of peptides derived from the purified protein allowed e xpressed sequence tag sequences to be identified that were assembled to yie ld a full-length composite sequence whose conceptual translation product co ntained the complete amino acid sequence of ovocalyxin-32. Data base search es revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebr al cortex and mast cells, and a skin protein, which is encoded by a retinoi c 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 lo calizes to the outer palisade layer, the vertical crystal layer, and the cu ticle of the eggshell, in agreement with its demonstration by Western blott ing at high levels in the uterine fluid during the termination phase of egg shell formation. Ovocalyxin-32 is therefore identified as a novel protein s ynthesized in the distal oviduct where hen eggshell formation occurs.