Carbohydrate isoelectric focussing: A new tool for the unprecedented separation of complex glycosaminoglycan structures.

在所有哺乳动物细胞的外部和细胞之间的空隙中，都有许多被称为糖胺聚糖（GAGs）的复杂多糖分子，它们控制着许多重要的生物过程。其中包括细胞生长和分裂，并涉及维持健康生物体和若干疾病过程，包括肿瘤、阿尔茨海默病、帕金森病和朊病毒疾病等的新血管生长。更好地了解这些分子的结构和活动是理解和更有效地干预这些过程的关键。gag与许多不同种类的蛋白质结合，包括那些负责细胞通讯的蛋白质。它们在不同的细胞类型中表达时具有微妙的不同序列，目前关于序列如何决定活性有很多争论。不幸的是，不可能从单个组织中分离出足够数量的许多gag（特别是最复杂的成员；硫酸肝素- HS）来进行有用的实验。从天然来源制备的gag不可避免地是混合物。此外，由于已分离的粗GAG材料，使用现有技术通常无法分离单个组分（特别是HS）。这对这一领域的研究构成了严重的障碍。目前使用的分离技术依赖于GAGs的两个物理特性；首先是凝胶渗透色谱法（GPC）可以得到它们的体积（近似于它们的长度），但相对粗糙。第二个性质是它们的负电荷，它可以变化。这是通过高性能强阴离子交换色谱（HPAEC）或常规凝胶电泳（PAGE）将粗GAG混合物或其片段分离成具有相似结构的分子池的基础。然而，许多gag或它们的片段具有相似的大小和电荷，因此这些技术代表了目前分离的极限。在这里，提出了一种方法，旨在利用到目前为止一直被忽视的附加特性。gag含有其他化学基团（胺），其中大多数被其他两个基团（n -硫酸盐或n -乙酰基）中的一个覆盖。我们的假设是，如果这些覆盖基团被移除，它将赋予分子正电荷（来自胺）和负电荷（来自酸和o -硫酸盐）。这将使一种称为等电聚焦（IEF）的强大技术得以使用。IEF广泛应用于蛋白质领域，它已经彻底改变了分离。双正电荷和负电荷允许分子聚焦在pH梯度已经建立的电泳凝胶上；每种不同的糖到达凝胶上不同的点。从凝胶中（常规）回收GAG并替换覆盖基团（有两种类型的覆盖胺的基团-都可以选择性地去除或用成熟的方法替换）。因此，将产生纯化的gag样品或其片段。GAGs的性质不同于大多数蛋白质，因此市售的IEF凝胶不适合。开发合适的凝胶（使用合适的凝胶材料和pH梯度）和建立最佳运行条件也将进行。这个项目的目标是确定IEF在gag上是可能的，提供前所未有的纯度样品，协助研究和进一步开发制药和生物技术制剂，最终使生物活性得到更好的理解，并使有效干预更接近于许多医疗情况。

On the outside of all mammalian cells and in the spaces between them, there are a number of complex polysaccharide molecules called glycosaminoglycans (GAGs), which are known to control many important biological processes. These include cell growth and division, and are implicated in the maintenance of healthy organisms and several disease processes including new blood vessel growth in tumours, Alzheimer's, Parkinson's and prion diseases among others. Gaining a better understanding of the structure and activities of these molecules is key to understanding and intervening more effectively in these processes. GAGs bind to many different categories of proteins, including those responsible for cell communications. They have subtly different sequences when expressed by different cell types and there is currently much debate as to how sequence determines activity. Unfortunately, it is not possible to isolate sufficient quantities of many GAGs (particularly the most complex member; heparan sulfate - HS) from individual tissues to undertake useful experiments. Preparations of GAGs from natural sources are inevitably mixtures. Furthermore, having isolated crude GAG material, it is often impossible to separate the individual components (esp. HS) using current techniques. This now presents a serious obstacle to research in this area. The separation techniques which are used at present rely on two physical characteristics of GAGs; the first is their volume (approximating to their length) is expolited by gel permeation chromatography (GPC), but is relatively crude. The second property is their negative charge, which can vary. This is the basis for separation of a crude GAG mixture or of its fragments) into pools of molecules with similar structures by either high performance strong anion exchange chromatography (HPAEC) or conventional gel electrophoresis (PAGE). However, many GAGs, or their fragments, have both similar size and charge and these techniques therefore represent the current limit of separation. Here, a method is proposed, which aims to exploit an additional feature that has, until now, been overlooked. GAGs contain other chemical groups (amines), most of which are covered by one of two other (N-sulfate or N-acetyl) groups. Our hypothesis is that, if these covering groups were removed, it would confer the molecules with both positive (from the amine) and negative charges (from acid and O-sulfate). This would enable a powerful technique called isoelectric focusing (IEF) to be used. IEF is widely used in the protein field , where it has revolutionised separation. The dual positive and negative charges allow the molecules to be focussed on an electrophoresis gel in which a pH gradient has been established; each subtly different sugar arriving at a different point on the gel. Following (routine) recovery of GAG from the gel and replacement of the covering groups (there are two types of groups covering the amine-both be selectively removed or replaced by well-established methods). Hence, purified samples of the GAGs, or their fragments would be produced. The properties of the GAGs are distinct from those of most proteins and commercially available IEF gels are not suitable. Development of suitable gels (using appropriate gel materials and pH gradients) and establishment of optimal running conditions will also be undertaken. This project will aim to establish that IEF is possible on the GAGs providing samples of unprecedented purity, assisting both research and the further development of pharmaceutical and biotechnological agents, ultimately allowing biological activity to be better understood and bringing effective intervention in a host of medical situations nearer.