| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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
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, 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
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