| Budget Amount *help |
¥3,090,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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