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In vitro folding of synthetic glycoprotein

合成糖蛋白的体外折叠

基本信息

批准号：
18580107
负责人：
HOJO Hironobu
金额：
$ 1.98万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580107/
关键词：
Glvcoorotein Glvcopeptide thioester Segment condensation Folding Chemical synthesis フォールデノング

项目摘要

Glycoproteins are engaged in many biological processes in living organism, such as cell growth, tumor metastasis and so on. To understand the precise role of glycoproteins, it is essential to obtain chemically homogeneous glyoproteins. To this end, we have been establishing a facile method for glycoprotein synthesis based on our thioester segment condensation method. A problem remained in this method is how to fold the synthetic glycoproteins. We sometimes observed misfolding of the synthetic glycoprotein in the case of the one having short carbohydrate chain. In this research, we aimed to synthesize glycoprotein having larger carbohydrate chain, such as complex type N-linked sugar, and fold it to a correct three dimensional structure.First, Fmoc-Asn carrying N-linked undecasaccharide unit was prepared for the solid-phase peptide synthesis (SPPS) of glycoprotein. However, we could not obtain enough amount for SPPS. Thus, we used commercially available Fmoc-Asn carring nonasaccharide unit for the synthesis of glyoprotein, which was not folded properly, when shorter sugar was attached. The result showed that even with the longer sugar chain, the glycoprotein did not fold in a particular conformation. We further synthesized nona-, mono- and nonglycosylated chemokine CCL27 composed of 95 amino acid residues using our strategy. The folding of this protein showed that the nona and monoglycosylated CCL27 had different disulfide bond pattern with other chemokines prepared previously. In contrast, nonglycosylated CCL27 had the same disulfide bonds with other chemokines. From these results, we concluded that glycosylation has significant effect on folding of polypeptide, regardless of the length of the carbohydrate chain.

糖蛋白在生物体内参与多种生物学过程，如细胞生长、肿瘤转移等。为了了解糖蛋白的确切作用，获得化学上均一的糖蛋白是必不可少的。为此，我们在硫酯链段缩合方法的基础上，建立了一种简便的糖蛋白合成方法。这种方法仍然存在的一个问题是如何折叠合成的糖蛋白。我们有时观察到合成的糖蛋白在有短碳水化合物链的情况下错误折叠。本研究旨在合成具有较大碳水化合物链的糖蛋白，如复合型N-联糖，并将其折叠成正确的三维结构。首先，制备了携带N-联十一糖单元的Fmoc-ASN，用于糖蛋白的固相肽合成。然而，我们无法获得足够的SPPS金额。因此，我们使用商业上可买到的携带非糖单元的Fmoc-ASN来合成糖蛋白，当连接较短的糖时，糖蛋白不能正确折叠。结果表明，即使糖链较长，糖蛋白也不会以特定的构象折叠。我们用我们的策略进一步合成了由95个氨基酸残基组成的非糖化、单糖化和非糖化趋化因子CCL27。折叠实验表明，NONA和单糖化的CCL27与已制备的其他趋化因子具有不同的二硫键模式。相反，非糖基化的CCL27与其他趋化因子具有相同的二硫键。根据这些结果，我们得出结论，糖基化对多肽的折叠有显著的影响，与碳水化合物链的长度无关。