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，并且假设高度有序的细胞糖基化受到所涉及的生物合成酶的独特底物特异性和容纳生物合成酶的高尔基体区室结构的调节。 PG 的糖胺聚糖 (GAG) 链是结构最复杂、生物学上最重要的聚糖之一。 GAG 结构，特别是硫酸乙酰肝素 (HS)，携带大量的生物信息。 HS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）信号复合物中发挥重要作用。 FGF 信号系统在发育生物学和癌症中非常重要。先前的研究表明 HS 以序列依赖性方式激活不同的 FGF7FGFR 复合物。这些研究表明 HS 存在离散的结构-活性关系 (SAR)。然而，HS-SAR 的测定极具挑战性，因为没有直接的方法来合成具有足够结构域大小和多种定义序列的 HS。 我们已经开始通过设计一种人工高尔基体细胞器来应对这一挑战，该细胞器能够控制合成结构明确的生物活性 GAG。这种人工细胞器基于新开发的数字微流体平台，旨在模拟天然高尔基体的基于流体膜的结构。这种人工高尔基体已经开始提供合成特定序列的 GAG 所需的精确时间和空间控制。我们设计的人工高尔基体适合计算机自动化，并且可以与在线电喷雾电离-液相色谱/质谱分析连接，促进GAG的高通量合成和分析。 该项目的五个具体目标是： 1. 使用人工高尔基体以受控和自动化的方式研究 HS 的生物合成； 2. 利用人工高尔基体合成1080个HS十糖结构亚型的有限组合文库； 3. 筛选这个有限的 HS 文库，以确定其通过 FGF 发出信号的能力； 4. 合成围绕这些命中扩展的 HS 十糖文库，以优化生物活性； 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，并建立其 SAR。这些研究的成果将是开发一个用于控制合成已定义聚糖文库的平台。将建立体外高通量筛选技术来研究这些聚糖的生物活性。使用这些方法，我们期望识别潜在的治疗性聚糖、它们的序列以及合成它们所需的特定酶合成参数。 公共健康相关性：拟议的工作通过开发人工高尔基体来合成硫酸乙酰肝素和硫酸乙酰肝素寡糖库来影响人类健康。这种人工高尔基体使用数字微流体平台在细胞微阵列上合成和筛选活性，以开发成纤维细胞生长因子信号传导的结构活性关系。