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A breakthrough to the microheterogeneity of the carbohydrate structure in glycoprotein by synthetic approach

合成方法突破糖蛋白碳水化合物结构微观异质性

基本信息

批准号：
16580093
负责人：
HOJO Hironobu
金额：
$ 1.92万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

Carbohydrates on proteins play various essential roles, such as cell-growth, differentiation, and tumor metastasis. However, the details of these functions are not understood at a molecular level, which mainly arises from the microheterogeneity at the carbohydrate portions. Especially, mucins retain a dense cluster of highly heterogeneous carbohydrates, which makes its functional analysis difficult.In this research, we developed a method to prepare glycopeptides carrying different carbohydrates at distinct amino acid residues to mimic the microheterogeneity of the natural glycoproteins, which will provide a basis to solve the problem arising from the microheterogeniety. First, we synthesized a glycopeptide derived from the consensus sequence of MUC5AC. During the solid-phase synthesis, Fmoc-Ser and Fmoc-Thr carrying core 8 carbohydrate were introduced at specific positions, giving the MUC5AC glycopeptide carrying two carbohydrates at different amino acid residues. In the second example … More , a novel approach to the glycopeptide carrying a couple of distinctively sialylated core 2 O-glycan was developed. The sequence derived from the leukosialin was divided into two shorter peptide segments. The N-terminal peptide thioester carrying one core 2 O-glycan was synthesized using the modified procedure for the Fmoc peptide thioester preparation. C-terminal peptide amide carrying a core 2 O-glycan was prepared by the conventional Fmoc method. The N- and C-terminal peptide was treated with different sialyl transferases giving two differently sialylated glycopeptides. These two segments were then condensed by the thioester method to give a glycopeptide carrying a couple of distinctively sialylated core 2 glycan. In the third example, a method to synthesize larger glycopeptide models of mucins was developed. The sequence derived from the repeating unit of MUC2, carrying 7 O-GalNAc moieties at specific positions, was prepared by the modified procedure for the Fmoc peptide thioester preparation. This peptide was then repeatedly condensed 6 times by the thioester method to obtain glycoprotein composed of 141 amino acid residues carrying 42 GalNAc moieties. At present, this is the largest glycoprotein prepared by the fully chemical procedure. The method can be used to synthesize mucin models carrying different carbohydrates at distinct positions.The synthetic heterogeneously glycosylated peptides prepared by the above procedures will be useful to understand the microheterogeniety of glycoproteins, especially of mucins. Less

蛋白质上的碳水化合物发挥着各种重要作用，例如细胞生长、分化和肿瘤转移。然而，这些功能的细节在分子水平上尚不清楚，这主要是由于碳水化合物部分的微观异质性引起的。特别是，粘蛋白保留了密集的高度异质性碳水化合物簇，这使得其功能分析变得困难。在本研究中，我们开发了一种制备在不同氨基酸残基上携带不同碳水化合物的糖肽的方法，以模拟天然糖蛋白的微观异质性，这将为解决微观异质性问题提供基础。首先，我们合成了源自MUC5AC共有序列的糖肽。在固相合成过程中，在特定位置引入携带核心8碳水化合物的Fmoc-Ser和Fmoc-Thr，得到在不同氨基酸残基处携带两种碳水化合物的MUC5AC糖肽。在第二个例子中，开发了一种携带几个独特唾液酸化核心 2 O-聚糖的糖肽的新方法。源自白唾液酸蛋白的序列被分成两个较短的肽片段。使用 Fmoc 肽硫酯制备的改进程序合成携带一个核心 2 O-聚糖的 N 末端肽硫酯。通过传统的Fmoc方法制备携带核心2O-聚糖的C端肽酰胺。 N-和C-末端肽用不同的唾液酸转移酶处理，得到两种不同唾液酸化的糖肽。然后通过硫酯法将这两个片段缩合，得到携带一对独特唾液酸化的核心2聚糖的糖肽。在第三个例子中，开发了一种合成更大的粘蛋白糖肽模型的方法。源自MUC2重复单元的序列在特定位置携带7个O-GalNAc部分，通过Fmoc肽硫酯制备的改进程序来制备。然后通过硫酯法将该肽重复缩合6次，得到由141个氨基酸残基组成、带有42个GalNAc部分的糖蛋白。这是目前通过全化学程序制备的最大的糖蛋白。该方法可用于合成在不同位置携带不同碳水化合物的粘蛋白模型。通过上述方法制备的合成异质糖基化肽将有助于理解糖蛋白，特别是粘蛋白的微观异质性。较少的