Toward an Artificial Golgi: Redesigning the Biological Activities of Heparan Sulfate on a Digital Microfluidic Chip
Toward an Artificial Golgi: Redesigning the Biological Activities of Heparan Sulfate on a Digital Microfluidic Chip
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DOI：
10.1021/ja903038d
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发表时间：
2009-08-12
影响因子：
15
通讯作者：
Linhardt, Robert J.
中科院分区：
文献类型：
--
作者：
Martin, Jeffrey G.;Gupta, Megha;Linhardt, Robert J.
利用数字微流体、重组酶技术和磁性纳米颗粒，我们创造了一个人工高尔基细胞器的功能原型。类似于天然高尔基体，它负责酶修饰固定在蛋白质上的糖胺聚糖，这种人工高尔基体酶修饰糖胺聚糖，特别是固定在磁性纳米颗粒上的硫酸肝素（HS）链。利用d -氨基葡萄糖基3- o -磺基转移酶将硫基团从x -磷酸腺苷5'-硫酸磷酸转移到固定化HS链上d -氨基葡萄糖残基的3-羟基上。经修饰后，经共聚焦显微镜检测，固定化HS纳米颗粒对荧光标记抗凝血酶III的亲和力增强。由于HS的生物合成涉及一系列专门的糖基转移酶、外聚酶和硫基转移酶，因此这种方法应该模拟HS在体内的合成。此外，我们的方法证明了研究多酶系统对最终糖聚糖产物结构的影响的可行性。
Using digital microfluidics, recombinant enzyme technology, and magnetic nanoparticles, we have created a functional prototype of an artificial Golgi organelle. Analogous to the natural Golgi, which is responsible for the enzymatic modification of glycosaminoglycans immobilized on proteins, this artificial Golgi enzymatically modifies glycosaminoglycans, specifically heparan sulfate (HS) chains immobilized onto magnetic nanoparticles. Sulfo groups were transferred from adenosine X-phosphate 5'-phosphosulfate to the 3-hydroxyl group of the D-glucosamine residue in an immobilized HS chain using D-glucosaminyl 3-O-sulfotransferase. After modification, the nanoparticles with immobilized HS exhibited increased affinity for fluorescently labeled antithrombin III as detected by confocal microscopy. Since the biosynthesis of HS involves an array of specialized glycosyl transferases, epimerase, and sulfotransferases, this approach should mimic the synthesis of HS in vivo. Furthermore, our method demonstrates the feasibility of investigating the effects of multienzyme systems on the structure of final glycan products for HS-based glycomic studies.