| Project/Area Number |
18570125
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Functional biochemistry
|
|---|
| Research Institution | Aichi University of Education |
|---|
Principal Investigator |
HABUCHI Osami Aichi University of Education, Education, Professor (90024067)
|
|---|
| Project Period (FY) |
2006 – 2007
|
| Project Status |
Completed (Fiscal Year 2007)
|
| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
|
|---|
| Keywords | chondroitin sulfate / sulfotransferases / gene knockout / chondroitin sulfate E / C6ST / GalNAc4S-6ST / N-glycan / bone marow derived mast cells / GalNAc4S-6ST / Tryptase / Thrombin / イカ軟骨 |
|---|
| Research Abstract |
1. We generated GalNAc4S-6ST KO mice. GalNAc4S-65T-/- mice lost synthetic activity of CS-E completely in every tissues. Bone marrow derived mast cells derived from the GalNAc4S-6ST-/- mice could not synthesize CS-E and showed decreased activity of tryptase.
2. We established stable clone expressing GalNAc4S-6ST from rat chondrosarcoma cell line. This clone synthesized and secreted proteoglycan containing CS-E. Chain length of CS produced by the clone was much shorter than the length of CS produced by the parent cells, suggesting that formation of GalNAc (4,6-SO4) residues by GalNAc4S-6ST may inhibit chain elongation.
3. We established a new method to synthesize a unique nonrreducing terminal sequence GalNAc(4,6-SO_4)-GlcA(2SO_4)-GalNAc(6SO_4), using 2OST and GalNAc4S-6ST from GalNAc(4SO_4)-GlcA-GalNAc(6SO_4).
4.We synthesized various C6ST-1 mutants devoid of N-glycosylation sites by the site-directed mutagenesis, and showed that N-glycans attached to C6ST-1 contributed to production of active enzyme, intracellular localization and substrate specificity of C6ST-I.
5.We cloned squid GalNAc4S-63T cDNA from the squid cartilage and prepared the recombinant enzyme. CS-E synthesized by the recombinant GalNAc4S-6ST inhibit thrombin in the antithrombin III-independent manner
|
