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间中，称为糖胺聚糖（GAG）的复杂多糖分子已知控制许多重要的生物过程。这些包括细胞生长和分裂，并涉及健康生物体的维持和几种疾病过程，包括肿瘤中的新血管生长、阿尔茨海默病、帕金森病和朊病毒病等。更好地了解这些分子的结构和生物活性是理解和更有效地干预这些过程的关键。GAG与许多不同类型的蛋白质结合，包括负责细胞通讯的蛋白质。当它们被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多争论。不幸的是，并不总是能够从单个组织中分离出足够量的许多GAG（特别是最复杂的成员，硫酸乙酰肝素- HS）用于有用的实验。天然来源的GAG制剂不可避免地是混合物。此外，在分离粗GAG材料后，直到最近，使用先前可用的技术难以分离单个组分（尤其是HS）。这对这一领域的研究构成了严重障碍。历史上，GAG分离技术依赖于GAG的两个物理特性;第一个是通过凝胶过滤色谱法（GFC）利用其流体动力学体积（近似于长度），尽管这相对粗糙。第二个属性是它们的负电荷，这是变化的。该策略将粗GAG混合物或其片段分离到具有相似结构的池中。然而，许多GAG或其片段具有相似的大小和电荷，因此这些技术代表了以前的分离极限。最近，申请人和合作者成功地展示了一种方法，该方法利用了一个额外的特征，该特征到目前为止一直被忽视。GAG含有其他化学基团（胺），其中大部分被两个其他（N-硫酸酯或N-乙酰基）基团之一覆盖。暴露这些基团赋予分子正电荷（来自所得胺）和负电荷（来自羧酸和O-硫酸酯基团）。这使得称为等电聚焦（IEF）的强大技术能够用于它们的分离（Holman &斯基德莫尔等人（2010）2：1550）。双重正电荷和负电荷（两性电解质）允许分子聚焦在电泳凝胶上，其中已经建立了pH梯度;每种细微不同的糖到达凝胶上的不同点。在从凝胶中（常规）回收GAG并替换覆盖基团（有两种类型的基团覆盖胺，两者都可以通过公认的方法选择性去除或替换）后。因此，现在可以生产GAG或其片段的纯化样品。IEF被广泛应用于蛋白质领域，在那里它已经彻底改变了分离，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，其具有稳定的、可再现的pH梯度的附加益处。开发合适的固定化pH梯度（IPG）凝胶和建立最佳运行条件的等电分离的糖胺酸是本建议的主要目的。还将研究将基于GAG的IEF方法转移到当前基于蛋白质的IEF系统的潜力，这无疑将促进广泛使用。这将使来自组织和其他生物样品的GAG的高分辨率IEF成为可能。这将最终使生物活动得到更好的理解，并使有效干预一系列医疗情况更近。

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.