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.
Center for Health and Environmental Risk Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba-city, Ibaraki, 305-8506, Japan.
Sci Rep. 2020 Oct 27;10(1):18335. doi: 10.1038/s41598-020-75444-4.
The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.
贝壳是一种由碳酸钙和有机基质组成的生物矿物，有机基质控制着碳酸钙晶体的生长。有机基质的主要成分是不溶性几丁质和蛋白质。通过使用酸或洗涤剂等试剂将其溶解，可以鉴定出各种蛋白质。然而，由于与几丁质形成了固体复合物，不溶性蛋白质仍然存在。本文中，我们使用巯基乙醇和胰蛋白酶从珍珠贝的珍珠层、棱柱层和铰合韧带中鉴定了这些蛋白质。大多数鉴定出的蛋白质都含有一个共同的富含蛋氨酸的区域。我们重点研究了这些蛋白质中的一种，即 NU-5，以研究其在贝壳形成中的功能。NU-5 的基因表达分析表明，NU-5 在套膜中高度表达，而 NU-5 的敲低阻止了珍珠层中霰石薄片的形成，这表明 NU-5 是形成珍珠层所必需的。动态光散射和圆二色性表明，重组的 NU-5 具有聚集活性，并在钙离子存在的情况下改变其二级结构。这些发现表明，富含蛋氨酸的不溶性蛋白质可能对支架形成很重要，支架形成是生物矿化的初始阶段。
