Department of Food and Nutrition, Faculty of Human Sciences and Design, Japan Women's University.
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology.
J Nutr Sci Vitaminol (Tokyo). 2022;68(4):284-293. doi: 10.3177/jnsv.68.284.
Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.
肠型碱性磷酸酶（IAP）在肠上皮细胞的刷状缘膜中高度表达，已知是肠道黏膜防御因子。在人类中，已分离出一个编码 IAP 的单一基因（ALPI），其转录产生两种选择性剪接的 mRNAs（aAug10 和 bAug10）。最近，我们发现维生素 D 可上调 Caco-2 细胞中两种类型的人 IAP 选择性剪接变体的表达。然而，mRNA 变体编码的蛋白质的功能差异仍不清楚。在本研究中，我们旨在进一步深入了解 IAP 同工型的特征和结构。为了分析 ALPI 基因翻译的蛋白质，我们构建了两种 cDNA 表达质粒（aAug10 和 bAug10），转染的细胞匀浆并测定碱性磷酸酶（ALP）活性。我们还设计了由 mRNA 变体（ALPI-aAug10 和 ALPI-bAug10）编码的蛋白质的同源建模 3D 结构。用 aAug10 质粒转染的 COS-1 细胞的 ALP 活性水平显著增加，而用 bAug10 质粒转染的细胞的 ALP 活性则无法检测到。同源建模 3D 结构显示，变体 bAug10 缺乏中央 N 端α-螺旋和活性位点附近对应于 ALPI-aAug10 的 Asp-42 的残基。这是关于人类 ALPI 基因选择性剪接转录变体的特征和结构的首次报道。进一步研究 aAug10 和/或 bAug10 mRNA 表达的调节可能会发现 IAP 的新的生理功能。
