In vitro folding of synthetic glycoprotein

糖蛋白参与了生物体内的许多生物学过程，如细胞生长、肿瘤转移等，要了解糖蛋白的确切作用，必须获得化学上均一的糖蛋白。为此，我们一直在基于硫酯片段缩合方法建立一种简便的糖蛋白合成方法。该方法中的一个问题是如何折叠合成的糖蛋白。我们有时会观察到合成糖蛋白在具有短糖链的情况下的错误折叠。本研究以合成具有较大糖链的糖蛋白（如复合型N-连接糖）为目标，首先制备了带有N-连接十一糖单元的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SPPS金额。因此，我们使用市售的携带九糖单元的Fmoc-Asn来合成糖蛋白，当连接较短的糖时，糖蛋白没有正确折叠。结果表明，即使是较长的糖链，糖蛋白也不会折叠成特定的构象。我们使用我们的策略进一步合成了由95个氨基酸残基组成的九糖基化、单糖基化和非糖基化趋化因子CCL 27。蛋白质折叠表明，Nona和单糖基化的CCL 27与先前制备的其他趋化因子具有不同的二硫键模式。相比之下，非糖基化的CCL 27与其他趋化因子具有相同的二硫键。从这些结果中，我们得出结论，糖基化对多肽的折叠有显着的影响，无论糖链的长度。

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.