Mano Hiroki, Kushioka Takuya, Kise Satoko, Nagao Chika, Iijima Ayano, Nishikawa Miyu, Ikushiro Shinichi, Yasuda Kaori, Matsuoka Sayuri, Sakaki Toshiyuki
Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
FANCL Research Institute, FANCL Corporation, 12-13 Kamishinano, Totsuka, Yokohama, Kanagawa 244-0806, Japan.
J Steroid Biochem Mol Biol. 2023 Mar;227:106233. doi: 10.1016/j.jsbmb.2022.106233. Epub 2022 Dec 9.
Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D (25(OH)D) and 1α,25-dihydroxyvitamin D (1α,25(OH)D) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D and 1α,25(OH)D are indicators of bone-related diseases such as rickets and osteoporosis. In this study, we have developed a novel LgBiT-LXXLL+LBD-SmBiT system using NanoLuc Binary Technology (NanoBiT), which has an emission intensity several times higher than that of the split-type firefly luciferase. Furthermore, by using genetic engineering techniques, we attempted to construct a novel system that can specifically detect 1α,25(OH)D. Because histidine residues at positions 305 and 397 play important roles in forming a hydrogen bond with a hydroxyl group at position C25 of 25(OH)D and 1α,25(OH)D, His305 and His397 were each substituted by other amino acids. Consequently, the three mutant VDRs, H305D, H397N, and H397E were equally useful to detect 1α,25(OH)D specifically. In addition, among the 58 variants of the LXXLL sequences, LPYEGSLLLKLLRAPVEE showed the greatest increase in luminescence upon the addition of 25(OH)D or 1α,25(OH)D. Thus, our novel system using NanoBiT appear to be useful for detecting native vitamin D or its derivatives.
此前，我们报道了一种FLucN-LXXLL+LBD-FLucC系统，该系统利用分裂萤火虫荧光素酶技术、维生素D受体（VDR）的配体结合域（LBD）以及VDR配体结合后与LBD相互作用的LXXLL序列来检测VDR配体。在体内，25-羟基维生素D（25(OH)D）和1α,25-二羟基维生素D（1α,25(OH)D）作为VDR配体与VDR结合，并调节骨相关基因的表达。因此，25(OH)D和1α,25(OH)D的量是佝偻病和骨质疏松症等骨相关疾病的指标。在本研究中，我们利用纳米荧光素酶二元技术（NanoBiT）开发了一种新型的LgBiT-LXXLL+LBD-SmBiT系统，其发射强度比分裂型萤火虫荧光素酶高几倍。此外，通过基因工程技术，我们试图构建一种能够特异性检测1α,25(OH)D的新型系统。由于305位和397位的组氨酸残基在与25(OH)D和1α,25(OH)D的C25位羟基形成氢键中起重要作用，His305和His397分别被其他氨基酸取代。因此，三种突变型VDR，即H305D、H397N和H397E，在特异性检测1α,25(OH)D方面同样有用。此外，在LXXLL序列的58个变体中，LPYEGSLLLKLLRAPVEE在添加25(OH)D或1α,25(OH)D后发光增加最为显著。因此，我们使用NanoBiT的新型系统似乎可用于检测天然维生素D或其衍生物。
