| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
The aim of the research is to find basic knowledge about the intracellular accumulation of biosynthetic enzymes of heparan sulfate/heparin. N-deacetlase/N-sulfotransforeases(NDSTs) had been used as a model to investigate whether their localization dependent on N-terminal sequences and interactions between their catalytic domains would be occurred.
First, relationship between the expression of NDST and the composition of disaccharide structure in heparan sulfate/heparin, using formerly established HEK 293 cells where one of mouse NDST was incorporated heterogeneously to be expressed by the induction. As a result, a portion of N-sulfation in disaccharides was increased in cells where the expression of either NDST1, 2 or 4 was induced, while significant changes have not been observed in that of NDST3. Those results are well correlated to their enzymatic properties in vitro.
Second, transient expression of fusion polypeptides composed of N-terminal region of either NDST3 or 4 and catalytic d
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omain of NDST1 had been examined in CHO-K1 cells to investigate where those polypeptides would be localized and whether their localization would be different from that of NDST1, resulting in the production different composition of heparan sulfate disaccharides. However, any significant changes in their structures have not been found when compared to negative control, indicating no evidence how fusion polypeptide contribute to the biosynthesis of heparan sulfate/heparin.
Third, transient expression of either NDTS4 or the mutated form of NDST1 or 2 to lower N-sulfotransferase activity had been examined in HEK 293 cells where mouse NDST3 was incorporated heterogeneously to be expressed by the induction to investigate interaction of NDSTs for effective biosynthesis of heparan sulfate/heparin. However, any significant changes in structures of disaccharides have not been found when compared to negative control.
Forth, fusion polypeptide of cytoplasmic region of NDST1 and GFP could be expressed in the cytoplasm. Less
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