Zhu Lingxiao, Wang Liying, Matsuura Akihiro, Zhang Mimin, Lu Peng, Iimura Kurin, Nagata Koji, Suzuki Michio
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Food Department, Department of Home Economics, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681, Japan.
Protein Expr Purif. 2021 Feb;178:105781. doi: 10.1016/j.pep.2020.105781. Epub 2020 Oct 31.
Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and β = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.
Pf-SCP是一种21千道尔顿的蛋白质，具有两个EF-手基序和一个磷酸化位点，从外套膜组织中鉴定出来，可与钙离子结合，并将钙成分从细胞转运到合浦珠母贝的贝壳中。为了揭示Pf-SCP钙结合活性的分子基础，我们在大肠杆菌中表达了全长Pf-SCP的重组蛋白。通过镍亲和层析和尺寸排阻层析纯化的重组Pf-SCP（rPf-SCP）在SDS-PAGE上呈现为单一条带。圆二色光谱表明，当rPf-SCP与钙离子和碳酸钙相互作用时，α-螺旋含量降低。蛋白质印迹和免疫染色验证了Pf-SCP在贝壳中的表达，且主要定位于外套膜上皮细胞。为了进一步了解钙离子和碳酸钙对Pf-SCP的结构和功能调节，我们进行了在钙离子存在下rPf-SCP的结晶实验。在钙离子存在下获得的rPf-SCP晶体对X射线的衍射分辨率达到1.8 Å。该晶体的空间群为C2，晶胞参数为a = 96.828 Å，b = 55.906 Å，c = 102.14 Å，β = 90.009°，根据马修斯系数的值估计，一个不对称单元中包含三个rPf-SCP分子。这些结果表明，Pf-SCP可能通过将钙离子集中在外套膜上皮细胞内并与碳酸钙分子相互作用，在钙离子运输和贝壳矿化中发挥作用。
