In vitro folding of synthetic glycoprotein

糖蛋白参与了生物体的许多生物过程，如细胞生长、肿瘤转移等。要了解糖蛋白的确切作用，必须获得化学上均匀的糖蛋白。为此，我们建立了一种基于我们的硫酯段缩合法的简便的糖蛋白合成方法。这种方法的一个问题是如何折叠合成的糖蛋白。在碳水化合物链短的情况下，我们有时观察到合成糖蛋白的错误折叠。在本研究中，我们的目标是合成具有更大碳水化合物链的糖蛋白，如复合型n链糖，并将其折叠成正确的三维结构。首先，制备了携带n链脱糖单元的Fmoc-Asn，用于糖蛋白的固相肽合成（SPPS）。然而，我们无法获得足够的金额用于SPPS。因此，我们使用市售的携带Fmoc-Asn的非糖单元来合成糖蛋白，当连接较短的糖时，糖蛋白不能正常折叠。结果表明，即使有较长的糖链，糖蛋白也不会以特定的构象折叠。我们进一步利用我们的策略合成了由95个氨基酸残基组成的非糖基化、单糖基化和非糖基化趋化因子CCL27。该蛋白的折叠结果表明，单糖基化CCL27与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。从这些结果，我们得出结论，无论碳水化合物链的长度如何，糖基化对多肽的折叠都有显著的影响。

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.