INVESTIGATION OF 1-DEOXYDIHYDROCERAMIDE BIOSYNTHESIS BY MAMMALIAN CELLS

哺乳动物细胞 1-脱氧二氢神经酰胺生物合成的研究

• 批准号: 8365570
• 负责人: ALFRED Harrison MERRILL
• 金额: $ 1.54万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365570
• 关键词: Alanine; Amino Acids; Anabolism; Animals; Biology; Categories; Cell Line; Cells; Ceramides; Coupled; Electrospray Ionization; Funding; Grant; High Pressure Liquid Chromatography; Incubated; Investigation; Mammalian Cell; Mass Spectrum Analysis; Medicine; Methods; National Center for Research Resources; Palmitoyl Coenzyme A; Physical condensation; Play; Principal Investigator; Pulsar; Regulation; Research; Research Infrastructure; Resolution; Resources; Role; Sampling; Serine; Source; Speed; Sphingolipids; Stable Isotope Labeling; Structure; Supplementation; Time; United States National Institutes of Health; base; clinically relevant; cost; interest; mass spectrometer; novel; overexpression; serine palmitoyltransferase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A novel sphingoid base, 1-deoxysphinganine, and its N-acyl-metabolites were recently discovered in studies of mammalian cells in culture and animals (Zitomer et al., J. Biol. Chem. 284:4786, 2009) and represent a novel new category of sphingolipid. 1-Deoxysphinganine was shown to arise from condensation of L-alanine with palmitoyl-CoA by cells in culture, and most is metabolized to 1-deoxydihydroceramides, rather than accumulating as the free sphingoid base. In anticipation of interest in these compounds, such as their possible elevation in clinically relevant samples, we have now developed a high-throughput and structure-specific method for analysis of 1-deoxysphingolipids by high performance liquid chromatography coupled to an electrospray ionization (ESI) QSTAR Pulsar i time-of-flight (TOF) mass spectrometer which combines, speed, sensitivity, high mass accuracy and high resolution. 1-Deoxydihydroceramides were extracted from a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) which was incubated with different stable isotope labeled amino acids, such as [3-13C]-L-Serine, [2,3-13C2]-L-Alanine, and [3-D3]-L-Alanine, and examined by LC-ESI MS/MS. These amino acid supplementations show that the novel 1-deoxysphingolipid arises from condensation of alanine with palmitoyl-CoA as evidenced by incorporation of [2,3-13C2]-L-alanine and [3-D3]-L-Alanine into 13C2-1-deoxydihydroceramides and D3-1-deoxydihydroceramides, respectively, by cells in culture. The results show that these underappreciated categories of bioactive sphingoid bases and "ceramides" that are likely to play important roles in cell regulation can be separated and detected by developed LC MS/MS methods.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 最近在对培养物和动物中的哺乳动物细胞的研究中发现了一种新的鞘氨醇碱，1-脱氧二氢鞘氨醇及其 N-酰基代谢物（Zitomer 等人，J. Biol. Chem. 284:4786, 2009），并代表了一种新的鞘氨醇碱。鞘脂的新类别。 1-脱氧二氢鞘氨醇是由培养细胞中 L-丙氨酸与棕榈酰辅酶 A 缩合产生的，并且大部分代谢为 1-脱氧二氢神经酰胺，而不是作为游离鞘氨醇碱积累。考虑到人们对这些化合物的兴趣，例如它们在临床相关样品中可能升高，我们现在开发了一种高通量和结构特异性的方法，通过高效液相色谱与电喷雾电离 (ESI) 联用来分析 1-脱氧鞘脂QSTAR Pulsar i 飞行时间 (TOF) 质谱仪集速度、灵敏度、高质量精度和高分辨率于一体。 1-脱氧二氢神经酰胺从稳定过表达丝氨酸棕榈酰转移酶的 Hek293 细胞系（SPT1/2 细胞）中提取，该细胞与不同的稳定同位素标记的氨基酸（例如 [3-13C]-L-丝氨酸、[2,3-13C2]）一起孵育-L-丙氨酸和[3-D3]-L-丙氨酸，并通过 LC-ESI MS/MS 进行检查。这些氨基酸补充表明，新型 1-脱氧鞘脂源自丙氨酸与棕榈酰辅酶 A 的缩合，如 [2,3-13C2]-L-丙氨酸和 [3-D3]-L-丙氨酸并入 13C2-1 中所证明的那样培养细胞分别产生-脱氧二氢神经酰胺和D3-1-脱氧二氢神经酰胺。结果表明，这些未被充分认识的生物活性鞘氨醇碱和“神经酰胺”可能在细胞调节中发挥重要作用，可以通过开发的 LC MS/MS 方法进行分离和检测。