An Artificial Golgi: Controlled GAG Synthesis and Screening

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由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的隔室结构调节。PG的糖胺聚糖（GAG）链是结构最复杂和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），携带大量生物信息。已发现HS在成纤维细胞生长因子（FGF）-成纤维细胞生长因子受体（FGFR）信号传导复合物中起重要作用。FGF信号系统在发育生物学和癌症中很重要。先前进行的研究表明，HS以序列依赖性方式激活不同的FGF/FGFR复合物。这些研究表明，有一个离散的结构活性关系（SAR）的HS。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各样的定义序列的HS。 我们已经开始解决这一挑战，设计一个人工高尔基体细胞器能够控制合成的结构定义，生物活性的糖胺聚糖。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流体膜结构。这种人工高尔基体已经开始提供精确的时间和空间控制所需的合成GAG的定义序列。我们所设计的人工高尔基体适合于计算机自动化，并且可以与在线电喷雾电离-液相色谱/质谱分析相结合，从而促进GAG的高通量合成和分析。 该项目的五个具体目标是：1。研究生物合成HS在一个控制和自动化的方式使用人工高尔基体; 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.筛选该有限的HS文库通过FGF发信号的能力; 4.合成围绕这些命中扩增的HS十糖文库以优化生物活性;以及5.确定HS十糖通过126种可能的FGFWFGFR信号传导复合物进行信号传导的特异性和效力，并建立它们的SAR这些研究的结果将是开发用于限定聚糖文库的受控合成的平台。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，我们希望确定潜在的治疗聚糖，它们的序列，以及它们的合成所需的特定酶促合成参数。 公共卫生相关性：所提出的努力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控平台在细胞微阵列上进行合成和筛选活性，以开发成纤维细胞生长因子信号传导的结构活性关系。

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.