Immobilized GAG derived ampholytes for enhanced low pH isoelectric focusing

在所有哺乳动物细胞的外部和它们之间的空隙中，被称为糖胺多聚糖(GAG)的复杂的多糖分子被认为控制着许多重要的生物过程。这包括细胞生长和分裂，并与维持健康的生物体和几个疾病过程有关，包括肿瘤、阿尔茨海默氏症、帕金森氏症和普恩病毒等疾病中的新血管生长。更好地了解这些分子的结构和生物活性是更有效地理解和干预这些过程的关键。GAGs与许多不同类别的蛋白质结合，包括那些负责细胞通讯的蛋白质。当它们由不同的细胞类型表达时，它们具有微妙的不同序列，目前关于序列如何决定活性存在很大争议。不幸的是，从单个组织中分离出足够数量的GAG(特别是最复杂的成员硫酸乙酰肝素-HS)用于有用的实验并不总是可能的。从天然来源制备的GAG不可避免地是混合物。此外，在分离出粗制的口香糖材料后，直到最近，还很难分离出单独的成分(特别是。HS)使用以前可用的技术。这现在对这一领域的研究构成了严重的障碍。从历史上看，Gag的分离技术依赖于Gag的两个物理特性；第一个是GFC利用其流体动力学体积(接近长度)，尽管这是相对粗糙的。第二个性质是它们的负电荷，负电荷是不同的。这一策略将粗制的GAG混合物或其碎片分离到具有相似结构的池中。然而，许多GAG或它们的碎片具有相似的大小和电荷，因此这些技术代表了以前的分离限制。最近，申请者和合作者成功地展示了一种方法，该方法利用了一个到目前为止一直被忽视的额外特征。GAG含有其他化学基团(胺)，其中大部分被另外两个基团(N-硫酸盐或N-乙酰)中的一个覆盖。发现这些基团会给分子带来正电荷(来自生成胺)和负电荷(来自羧酸和O-硫酸盐基团)。这使得一种名为等电聚焦(IEF)的强大技术能够用于分离它们(Holman和Skinmore等人)。(2010)2：1550)。双正负电荷(两性离子)使分子聚焦在已建立pH梯度的电泳胶上；每个细微不同的糖到达凝胶上的不同点。随后(常规)从凝胶中回收GAG并替换覆盖基团(有两种类型的覆盖胺的基团，都可以选择性地移除或用成熟的方法替换)。因此，现在可以生产出这些恶作剧的纯化样本或它们的碎片。离子交换膜被广泛应用于蛋白质领域，它使分离发生了革命性的变化，尽管这主要是由于商业上可获得的固定化pH梯度凝胶的发展，这种凝胶具有稳定、可重复的pH梯度的额外好处。这些凝胶的性质与大多数蛋白质不同，商业上可用的IEF凝胶是不合适的。开发合适的固定化pH梯度(IPG)凝胶和建立GAG等电分离的最佳运行条件是这一建议的主要目的。还将研究将基于GAG的IEF方法转移到当前基于蛋白质的IEF系统的可能性，这无疑将促进广泛使用。这将使来自组织和其他生物样本的GAG的高分辨率IEF成为可能。这最终将使生物活动得到更好的理解，并使对一系列医疗状况的有效干预变得更近。

On the outside of all mammalian cells and in the spaces between them, complex polysaccharide molecules called glycosaminoglycans (GAGs) 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. Obtaining a better insight into the structure and biological 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 always possible to isolate sufficient quantities of many GAGs (particularly the most complex member, heparan sulfate - HS) from individual tissues for useful experimentation. Preparations of GAGs from natural sources are inevitably mixtures. Furthermore, having isolated crude GAG material, it was until recently, difficult to separate the individual components (esp. HS) using previously available techniques. This now presents a serious obstacle to research in this area. Historically, GAG separation techniques relied on two physical characteristics of GAGs; the first is their hydrodynamic volume (approximating to length) is exploited by gel filtration chromatography (GFC), although this is relatively crude. The second property is their negative charge, which varies. This strategy separates crude GAG mixtures, or fragments thereof, into pools with similar structures. However, many GAGs, or their fragments, have both similar size and charge and these techniques therefore represented the previous limit of separation.Recently, the applicant and collaborator demonstrated successfully a method that exploited 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. Uncovering these groups confers the molecules with both positive (from the resultant amine) and negative charges (from carboxylic acid and O-sulfate groups). This has enabled a powerful technique called isoelectric focusing (IEF) to be used for their separation (Holman & Skidmore et al. (2010) 2:1550).The dual positive and negative charges (ampholytes) 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 can be selectively removed or replaced by well-established methods). Hence, purified samples of the GAGs, or their fragments many now be produced. IEF is widely used in the protein field, where it has revolutionised separations although this has mainly been due to the development of commercially available immobilised pH gradient gels which have the added benefit of a stable, reproducible pH gradient.The properties of the GAGs are distinct from those of most proteins and commercially available IEF gels are unsuitable. The development of suitable immobilised pH gradient (IPG) gels and the establishment of optimal run conditions for the isoelectric separation of GAGs is the main aim of this proposal. The potential to transfer GAG based IEF methodologies to current protein based IEF systems will also be investigated, which will undoubtedly facilitate widespread use. This will enable the high resolution IEF of GAGs from tissue and other biological samples. This will ultimately allow biological activities to be better understood and bring effective intervention in a host of medical situations nearer.