Hirabayashi J, Arata Y, Kasai K
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, 199-0195, Japan.
Proteomics. 2001 Feb;1(2):295-303. doi: 10.1002/1615-9861(200102)1:2<295::AID-PROT295>3.0.CO;2-C.
Glycans play a central role as potential mediators between complex cell societies, because all living organisms consist of cells covered with diverse carbohydrate chains reflecting various cell types and states. However, we have no idea how diverse these carbohydrate chains actually are. The main purpose of this article is to persuade life scientists to realize the fundamental importance of taking some action by becoming involved in "glycomics". "Glycome" is a term meaning the whole set of glycans produced by individual organisms, as the third bioinformative macromolecules to be elucidated next to the genome and proteome. Here a basic strategy is presented. The essence of the project includes the following: (a) glycopeptides, but not glycans released from their core proteins, are targeted for linkage to genome databases; (b) Caenorhabditis elegans is used as the first model organism for this project, since its genome project has already been completed; (c) four essential attributes are adopted to characterize each glycopeptide: (i) cosmid identification number (ID), (ii) molecular weight (M(r)), (iii) retention (Rs) of pyridylaminated (PA) oligosaccharides in 2-D mapping, and (iv) dissociation constants (Kd's) of PA-oligosaccharides for a set of lectins. Thus, the obtained ID, M(r), R and Kd's construct the glycome database, which will be open as the previous genome and proteome databases. For the project to proceed the "glyco-catch" method is proposed, where a group of target glycopeptides are captured by means of lectin-affinity chromatography after protease digestion. Already glycopeptides from asialofetuin and ovalbumin were successfully captured by galectin-agarose and Con A-agarose, respectively. Further, to examine the practical validity of the method, we extracted membrane proteins from C. elegans with 1% Triton X-100, and isolated specific glycopeptides by use of the same galectin column. One of the glycopeptides was successfully identified in the C. elegans genome database. Finally, for determination of Kd between glycopeptides and lectins, a recently reinforced frontal affinity chromatography (FAC) is proposed as an alternative to define glycan structures in place of determining every covalent structure.
聚糖作为复杂细胞群落之间潜在的介质发挥着核心作用，因为所有生物都是由覆盖着反映各种细胞类型和状态的多样碳水化合物链的细胞组成。然而，我们并不清楚这些碳水化合物链实际上有多多样。本文的主要目的是说服生命科学家认识到通过参与“糖组学”采取一些行动的根本重要性。“糖组”是一个术语，指个体生物体产生的聚糖的完整集合，是继基因组和蛋白质组之后要阐明的第三种生物信息大分子。这里提出了一个基本策略。该项目的核心包括以下几点：（a）靶向糖肽，而非从其核心蛋白释放的聚糖，用于与基因组数据库建立联系；（b）秀丽隐杆线虫被用作该项目的第一个模式生物，因为其基因组项目已经完成；（c）采用四个基本属性来表征每个糖肽：（i）黏粒识别号（ID），（ii）分子量（M(r)），（iii）二维图谱中吡啶氨基化（PA）寡糖的保留时间（Rs），以及（iv）一组凝集素的PA-寡糖解离常数（Kd's）。因此，所获得的ID、M(r)、Rs和Kd's构建了糖组数据库，该数据库将像之前的基因组和蛋白质组数据库一样开放。为了推进该项目，提出了“糖捕获”方法，即在蛋白酶消化后，通过凝集素亲和色谱法捕获一组目标糖肽。来自去唾液酸胎球蛋白和卵清蛋白的糖肽已分别成功被半乳糖凝集素-琼脂糖和伴刀豆球蛋白A-琼脂糖捕获。此外，为了检验该方法的实际有效性，我们用1% Triton X-100从秀丽隐杆线虫中提取膜蛋白，并使用相同的半乳糖凝集素柱分离特定的糖肽。其中一个糖肽在秀丽隐杆线虫基因组数据库中成功得到鉴定。最后，为了测定糖肽与凝集素之间的Kd，提出了一种最近改进的前沿亲和色谱法（FAC），作为一种替代方法来定义聚糖结构，而不是确定每一个共价结构。
