Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
J Struct Biol. 2020 Oct 1;212(1):107594. doi: 10.1016/j.jsb.2020.107594. Epub 2020 Jul 29.
The shells of the Pacific oyster Crassostrea gigas contain calcite crystals with three types of microstructures: prismatic, chalky, and foliated layers. Many shell matrix proteins were annotated from the shells of C. gigas; however, it is unclear which SMPs play important roles in their shell mineralization. The matrix proteins have never been reported from the chalky layer. In this study, we identified a chalky layer-derived EGF-like domain-containing protein (CgELC) from the chalky layer of C. gigas shells. The gene sequence of the CgELC was encoded under CGI_ 10,017,544 of the C. gigas genome database. Only peptide fragments in the N-terminal region of CGI_ 10,017,544 were detected by LC-MS/MS analyses, suggesting that CGI_ 10,017,544 was digested at the predicted protease digestion dibasic site by post-translational modification to become a mature CgELC protein. We produced three types of CgELC recombinant proteins, namely, the full length CgELC, as well as the N-terminal and C-terminal parts of CgELC (CgELC-N or -C, respectively), for in vitro crystallization experiments. In the presence of these recombinant proteins, the aggregation of polycrystalline calcite was observed. Some fibrous organic components seemed to be incorporated into the calcite crystals in the presence of the r-CgELC protein. We also noted different features in the crystallization between CgELC-N and CgELC-C; some crystals were inhibited crystal plane formation and contained many columnar prisms inside the crystals (CgELC-N) and formed numerous holes on their surfaces (CgELC-C). These results suggest that CgELC is involved in crystal aggregation and incorporated into calcite crystals.
太平洋牡蛎（Crassostrea gigas）的壳内含有方解石晶体，具有三种微观结构：棱柱层、霰石层和叶片层。从太平洋牡蛎的壳中注释了许多壳基质蛋白；然而，尚不清楚哪些 SMP 在其壳矿化中起重要作用。基质蛋白从未在霰石层中报道过。在这项研究中，我们从太平洋牡蛎壳的霰石层中鉴定出一种来源于霰石层的表皮生长因子样结构域包含蛋白（CgELC）。CgELC 的基因序列编码在太平洋牡蛎基因组数据库的 CGI_10,017,544 之下。LC-MS/MS 分析仅检测到 CGI_10,017,544 的 N 端区域的肽片段，这表明 CGI_10,017,544 通过翻译后修饰在预测的蛋白酶消化双碱基位点被消化，成为成熟的 CgELC 蛋白。我们产生了三种类型的 CgELC 重组蛋白，即全长 CgELC 以及 CgELC 的 N 端和 C 端部分（分别为 CgELC-N 或 CgELC-C），用于体外结晶实验。在这些重组蛋白的存在下，观察到多晶方解石的聚集。在 r-CgELC 蛋白的存在下，一些纤维状有机成分似乎被掺入到方解石晶体中。我们还注意到 CgELC-N 和 CgELC-C 之间结晶的不同特征；一些晶体抑制了晶体平面的形成，并且在晶体内部含有许多柱状棱柱（CgELC-N）并且在其表面形成许多孔（CgELC-C）。这些结果表明 CgELC 参与晶体聚集并掺入方解石晶体中。
