Cloning and Characterization of Xenopus p26, an S100-like Calcium-binding protein in Xenopus Egg

Abstract

The early egg fertilization process requires a change in the intracellular calcium concentration. To understand the calcium-dependent molecular mechanisms of fertilization of eggs, we isolated a 26 kDa Ca²⁺-binding protein from Xenopus eggs, a homologue of Rana Catesbeiana p26 (renamed from p26olf) isolated from the olfactory epithelium. The primary structure of Xenopus p26 shows comparatively high amino acid identity (61%) to Rana p26, and consists of two S100-like regions aligned in tandem as seen in Rana p26. By ⁴⁵Ca blot analysis and flow dialysis experiments using ⁴⁵Ca, p26 was found to bind ∼4 Ca²⁺ with an apparent Kd value of ∼9.5 μM. Genomic Southern analysis implicated that Xenopus p26 is a unique orthologue of Rana. Northern blot analysis showed that Xenopus p26 is expressed in Xenopus eggs and also in other tissues. Immunohistochemical study revealed that Xenopus p26 is localized prominently in the cytoplasm of the cortex of both the animal and the vegetal hemisphere of Xenopus eggs. Blot overlay analysis showed that several egg proteins bind to Xenopus p26 in a Ca²⁺-dependent manner, indicating the presence of target proteins of Xenopus p26. These results indicated that Xenopus p26 is a novel S100-like protein in eggs and could be involved in the early Ca²⁺-dependent event during (or after) fertilization. [J Physiol Sci. 2006;56 Suppl:S220]