Novel Approaches to Access GPIs and GPI-Anchored Proteins for the Study of GPI An

获取 GPI 和 GPI 锚定蛋白用于 GPI An 研究的新方法

基本信息

  • 批准号:
    8324038
  • 负责人:
  • 金额:
    $ 29.37万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2009
  • 资助国家:
    美国
  • 起止时间:
    2009-09-30 至 2014-01-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Many surface proteins and glycoproteins are anchored to cell membranes by glycosylphosphatidylinositols (GPIs), and GPI-anchored proteins and glycoproteins play an important role in various biological processes. To investigate these processes and to explore GPI anchorage, it is necessary to have access to homogenous and structurally defined GPIs, GPI derivatives, and GPI-anchored proteins and glycoproteins. This proposal aims to develop a practical method for the synthesis of these important molecules and study GPI-anchored proteomics by means of synthetic GPIs. The proposal has three specific aims. Specific Aim 1 is to develop a general strategy for the synthesis of natural GPIs and various important GPI derivatives bearing functionalities such as alkene, alkyne, ester, thiol, and sulfide. This research plans to use the p-methoxybenzyl (PMB) group as a global and permanent protecting group in the synthesis to assure that the final deprotection can be achieved selectively in the presence of a variety of functionalities as mentioned. Specific Aim 2 is to develop a practical method for the chemical synthesis of natively linked GPI-anchored peptides/glycopeptides or proteins/glycoproteins. The fundamental synthetic design is to prepare the GPI and peptide/glycopeptide segments separately and then link them together via a mild and chemoselective reaction. For this purpose, synthetic GPIs will be modified to bear a hydroxylamine or methoxylamine group at the GPI glycan non-reducing end, and peptides will be modified to bear an 1-ketoacid functionality at the C-terminus. Subsequently, the two segments will be coupled by Bode's ligation method under mild conditions. Specific Aim 3 is to develop a feasible method for rapid isolation and identification of GPI-anchored proteins and glycoproteins and use it to study surface GPI anchorage. For this purpose, the endoplasmic reticulum (ER) membranes of mutant cells lacking GPI biosynthesis functionality will be isolated and used to react with synthetic biotinylated GPIs. Since these ER membranes contain both GPI transamidases, the enzymes that catalyze GPI addition to proteins, and all nascent proteins destined for GPI anchorage, their reaction with biotinylated GPIs will result in the biotinylation of all GPI-anchored proteins. This will create a cell-free system for obtaining the entire complement of GPI-anchored proteins in the biotinylated form. Thereafter, GPI-anchored proteins will be easily isolated with a streptavidin column and analyzed by MS and other methods established for proteomic studies. The broad impact of this project: An effective and widely useful method for the synthesis of homogenous and structurally defined GPIs and GPI-anchored peptides/glycopeptides/proteins is essential for the study and understanding of GPI anchorage and GPI-anchored molecules. Systematic analysis of GPI-anchored proteins on cells will provide important information about the profiles of cell GPI anchorage. This information can help identify GPI-anchored protein markers associated with certain diseases, which can be used as new molecular targets for the design and development of more effective diagnostic techniques and disease treatments. PUBLIC HEALTH RELEVANCE: This proposal aims to develop a practical synthetic method for glycosylphosphatidylinositols (GPIs) and GPI- anchored peptides/glycopeptides or proteins/glycoproteins that play pivotal roles in many biological processes and to develop a practical method for identifying and analyzing GPI-anchored proteins/glycoproteins expressed by cells. This research is important for the study and understanding of the functions of GPI anchors and GPI- anchored molecules. Systematic analysis of GPI-anchored proteins on cells will provide important information that can help identify disease-associated GPI-anchored protein markers that are useful molecular targets for the design and development of more effective diagnostic techniques and disease treatments.
描述(由申请人提供):许多表面蛋白和糖蛋白通过糖基磷脂酰肌醇(GPIs)锚定在细胞膜上,GPIs锚定蛋白和糖蛋白在各种生物过程中发挥重要作用。为了研究这些过程并探索GPI锚定,有必要获得同质和结构定义的GPI,GPI衍生物和GPI锚定蛋白和糖蛋白。本研究旨在开发一种合成这些重要分子的实用方法,并通过合成GPIs来研究GPI锚定蛋白质组学。该提案有三个具体目标。 具体目标1是开发用于合成天然GPIs和各种重要的GPIs衍生物的一般策略,所述GPIs衍生物具有诸如烯烃、炔、酯、硫醇和硫化物的官能团。本研究计划在合成中使用对甲氧基苄基(PMB)基团作为全局和永久的保护基团,以确保在所提到的各种官能团存在下可以选择性地实现最终的脱保护。 具体目标2是开发用于天然连接的GPI锚定肽/糖肽或蛋白质/糖蛋白的化学合成的实用方法。基本的合成设计是分别制备GPI和肽/糖肽片段,然后通过温和的化学选择性反应将它们连接在一起。为此目的,合成的GPI将被修饰以在GPI聚糖非还原末端携带羟胺或甲氧基胺基团,并且肽将被修饰以在C-末端携带1-酮酸官能团。随后,在温和条件下通过Bode连接法偶联两个片段。 具体目标3是开发一种可行的快速分离和鉴定GPI锚定蛋白和糖蛋白的方法,并用于研究表面GPI锚定。为此,将分离缺乏GPI生物合成功能的突变细胞的内质网(ER)膜,并用于与合成的生物素化GPI反应。由于这些ER膜含有GPI转酰胺酶(催化GPI添加到蛋白质的酶)和所有用于GPI锚定的新生蛋白质,它们与生物素化GPI的反应将导致所有GPI锚定蛋白质的生物素化。这将产生用于获得生物素化形式的GPI锚定蛋白的完整补体的无细胞系统。此后,GPI锚定的蛋白质将很容易地用链霉亲和素柱分离,并通过MS和其他为蛋白质组学研究建立的方法进行分析。 该项目的广泛影响:一种有效且广泛有用的方法,用于合成同质和结构定义的GPIs和GPI锚定肽/糖肽/蛋白质,对于研究和理解GPI锚定和GPI锚定分子至关重要。对GPI锚定蛋白在细胞上的系统分析将为了解细胞GPI锚定特性提供重要信息。这些信息可以帮助识别与某些疾病相关的GPI锚定蛋白标记物,这些标记物可以用作设计和开发更有效的诊断技术和疾病治疗的新分子靶点。 公共卫生相关性:本提案旨在开发在许多生物过程中起关键作用的糖基磷脂酰肌醇(GPIs)和GPI锚定肽/糖肽或蛋白/糖蛋白的实用合成方法,并开发用于鉴定和分析由细胞表达的GPI锚定蛋白/糖蛋白的实用方法。本研究对于深入研究GPI锚和GPI锚定分子的功能具有重要意义。对细胞上GPI锚定蛋白的系统分析将提供重要的信息,可以帮助识别疾病相关的GPI锚定蛋白标记物,这些标记物是设计和开发更有效的诊断技术和疾病治疗的有用分子靶点。

项目成果

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Zhongwu Guo其他文献

Zhongwu Guo的其他文献

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{{ truncateString('Zhongwu Guo', 18)}}的其他基金

Brain glycosphingolipids and Alzheimer's disease
脑鞘糖脂与阿尔茨海默病
  • 批准号:
    10738379
  • 财政年份:
    2023
  • 资助金额:
    $ 29.37万
  • 项目类别:
Development of New Vaccine Adjuvants
新型疫苗佐剂的开发
  • 批准号:
    10636922
  • 财政年份:
    2022
  • 资助金额:
    $ 29.37万
  • 项目类别:
Development of New Vaccine Adjuvants
新型疫苗佐剂的开发
  • 批准号:
    10480226
  • 财政年份:
    2022
  • 资助金额:
    $ 29.37万
  • 项目类别:
Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes
GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究
  • 批准号:
    9902533
  • 财政年份:
    2019
  • 资助金额:
    $ 29.37万
  • 项目类别:
Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes
GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究
  • 批准号:
    10371134
  • 财政年份:
    2019
  • 资助金额:
    $ 29.37万
  • 项目类别:
Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes
GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究
  • 批准号:
    10584557
  • 财政年份:
    2019
  • 资助金额:
    $ 29.37万
  • 项目类别:
New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics
获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法
  • 批准号:
    8628408
  • 财政年份:
    2009
  • 资助金额:
    $ 29.37万
  • 项目类别:
New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics
获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法
  • 批准号:
    8989113
  • 财政年份:
    2009
  • 资助金额:
    $ 29.37万
  • 项目类别:
Novel Approaches to Access GPIs and GPI-Anchored Proteins for the Study of GPI An
获取 GPI 和 GPI 锚定蛋白用于 GPI An 研究的新方法
  • 批准号:
    7938107
  • 财政年份:
    2009
  • 资助金额:
    $ 29.37万
  • 项目类别:
New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics
获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法
  • 批准号:
    9027236
  • 财政年份:
    2009
  • 资助金额:
    $ 29.37万
  • 项目类别:

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乙酰胆碱酯酶抑制剂对患有轻至中度阿尔茨海默病的老年人骨代谢和骨折危险因素的影响
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