An Artificial Golgi: Controlled GAG Synthesis and Screening

人工高尔基体：受控 GAG 合成和筛选

• 批准号: 7945295
• 负责人: Jonathan S. Dordick
• 金额: $ 29.49万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7945295
• 关键词: Abbreviations; Acetylgalactosamine; Acetylglucosamine; Address; Antithrombin III; Automation; Binding; Biological; Bromides; Cell Communication; Cell Proliferation; Cells; Chondroitin Sulfates; Communication; Complex; Computers; Deacetylase; Dermatan Sulfate; Detection; Development; Developmental Biology; Electrospray Ionization; Enzymes; Extracellular Matrix; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Glucosamine; Glucuronates; Glutamate Carboxypeptidase II; Glycosaminoglycans; Golgi Apparatus; Green Fluorescent Proteins; Health; Heparan Sulfate Biosynthesis; Heparin; Heparitin Sulfate; High Pressure Liquid Chromatography; Housing; Human; Hyaluronic Acid; In Vitro; Keratan Sulfate; Libraries; Liquid Chromatography; Liquid substance; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Methodology; Methods; Methylmethacrylate; Microfluidics; Mus; Oligosaccharides; Organelles; Outcome Study; Personal Computers; Phosphate Buffer; Play; Polysaccharides; Post-Translational Protein Processing; Printing; Process; Proteins; Proteoglycan; Receptor Signaling; Role; Saline; Screening procedure; Signal Transduction; Simulate; Sodium Dodecyl Sulfate; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Structure; Structure-Activity Relationship; Substrate Specificity; Surface Plasmon Resonance; System; Techniques; Therapeutic; Uridine Diphosphate; base; cell growth; cofactor; combinatorial; design; digital; epimerase; glycoprotein biosynthesis; glycosylation; high throughput screening; liquid chromatography mass spectrometry; peripheral blood; polysulfated glycosaminoglycan; protein complex; proto-oncogene protein kfgf; public health relevance; research study; sulfotransferase; tandem mass spectrometry

DESCRIPTION (provided by applicant): The Golgi is the organelle responsible for the posttranslational modification of proteins. Glycosylation, the most prominent posttranslational modification, results in the biosynthesis of glycoproteins and proteoglycans (PGs). Glycosylation is not template driven and it is hypothesized that highly ordered cellular glycosylation is regulated by the unique substrate specificities of the biosynthetic enzymes involved and the Golgi's compartmental structures housing the biosynthetic enzymes. The glycosaminoglycan (GAG) chains of PGs are among the most structurally complex and biologically important glycans. GAG structure, particularly heparan sulfate (HS), carries immense amounts of biological information. HS has been found to play an important role in fibroblast growth factor (FGF)-fibroblast growth factor receptor (FGFR) signaling complexes. The FGF signaling system is important in developmental biology and in cancer. Previous studies performed have shown that HS activates different FGF7FGFR complexes in a sequence dependent manner. These studies suggest that there is a discrete structure-activity relationship (SAR) for HS. The determination of HS-SAR, however, is extremely challenging, as there are no straightforward methods for the synthesis of HS having sufficient domain size and a wide variety of defined sequences. We have begun to address this challenge by designing an artificial Golgi organelle capable of the controlled synthesis of structurally defined, bioactive GAGs. This artificial organelle is based on a newly developed digital microfluidic platform designed to simulate the fluid membrane-based structure of the natural Golgi. This artificial Golgi has begun to provide the precise temporal and spatial control needed for the synthesis of GAGs of defined sequences. The artificial Golgi we have designed is amenable to computer automation and can be interfaced with on-line electrospray ionization-liquid chromatography/mass spectrometry analysis, facilitating high-throughput synthesis and analysis of GAGs. The five specific aims of this project are: 1. Investigate the biosynthesis of HS in a controlled and automated fashion using an artificial Golgi; 2. Synthesize a limited combinatorial library of 1080 HS decasaccharide structural subtypes using the artificial Golgi; 3. Screen this limited HS library for its ability to signal through FGF; 4. Synthesize an HS decasaccharide library expanded around these hits to optimize bioactivity; and 5. Determine the specificity and potency of the HS decasaccharides to signal through the 126 possible FGFWFGFR signaling complexes and establish their SAR The outcome of these studies will be the development of a platform for the controlled synthesis of defined glycan libraries. In vitro high-throughput screening techniques will be established for investigating the biological activity of these glycans. Using these methodologies, we expect to identify potential therapeutic glycans, their sequences, and the specific enzymatic synthesis parameters required for their synthesis. PUBLIC HEALTH RELEVANCE: The proposed effort impacts human health by developing an artificial Golgi to synthesize a library of heparan sulfates and heparan sulfate oligosaccharides. This artificial Golgi uses a digital microfluidic platform for synthesis and screen activity on a cell microarray to develop a structure activity relationship for fibroblast growth factor signaling.

描述（由申请人提供）：高尔基体是负责蛋白质翻译后修饰的细胞器。糖基化是最重要的翻译后修饰，导致糖蛋白和蛋白聚糖（pg）的生物合成。糖基化不是模板驱动的，据推测，高度有序的细胞糖基化是由所涉及的生物合成酶的独特底物特异性和高尔基体容纳生物合成酶的室室结构调节的。pg的糖胺聚糖（GAG）链是结构最复杂和生物学上重要的聚糖之一。GAG结构，特别是硫酸肝素（HS），携带着大量的生物信息。HS已被发现在成纤维细胞生长因子(FGF)-成纤维细胞生长因子受体（FGFR）信号复合物中起重要作用。FGF信号系统在发育生物学和癌症中都很重要。先前的研究表明，HS以序列依赖的方式激活不同的FGF7FGFR复合物。这些研究表明HS具有离散的构效关系（SAR）。然而，HS- sar的测定是极具挑战性的，因为没有直接的方法来合成具有足够结构域大小和各种定义序列的HS。我们已经开始通过设计一种人工高尔基细胞器来解决这一挑战，该细胞器能够控制合成结构明确的生物活性gag。这种人工细胞器是基于一种新开发的数字微流控平台，旨在模拟天然高尔基体的流体膜结构。这种人工高尔基体已经开始为合成特定序列的gag提供精确的时间和空间控制。我们设计的人工高尔基体可用于计算机自动化，并可与在线电喷雾电离-液相色谱/质谱分析相结合，便于高通量合成和分析gag。该项目的五个具体目标是：1。利用人工高尔基体在受控和自动化的方式下研究HS的生物合成；2. 利用人工高尔基体合成1080个HS十糖结构亚型有限组合文库3. 筛选这个有限的HS库通过FGF发出信号的能力；4. 合成一个围绕这些hit扩展的HS十糖库以优化生物活性；和5。确定HS十糖通过126种可能的FGFWFGFR信号复合物发出信号的特异性和效力，并建立它们的SAR。这些研究的结果将是开发一个用于控制合成定义聚糖库的平台。为研究这些多糖的生物活性，将建立体外高通量筛选技术。使用这些方法，我们期望确定潜在的治疗聚糖，它们的序列，以及合成它们所需的特定酶合成参数。