| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
|
|---|
| Research Abstract |
(1) We developed an enzymatic method capable of synthesizing CS-E in vitro. The enzymatically synthesized CS-E was bound strongly to midkine.
(2) We found that a unique nonreducing terminal structure, GalNAc(4-SO_4)-GlcA(2SO_4)-GalNAc(6SO_4), in chondroitin sulfate and that this structure was converted by GalNAc4S-6ST to a highly sulfated sequence, (GalNAc(4,6-SO_4)-GlcA(2SO_4)-GalNAc(6SO_4). The highly sulfated nonreducing sequence was found in chondroitin sulfated synthesized by the cultured chick chondrocytes, indicating that this structure appears to be present ubiquitously in the cartilage chondroitin sulfate.
(3) N-linked oligosaccharides attached to C4ST were found to be required for the enzyme activity. Deletion of the oligosaccharide located to the C-terminal region of C4ST resulted not only in drastic decrease in the activity but also in the alteration of glycosylation pattern.
(4) Desulfated dermatan sulfate was efficiently sulfated by the recombinant C6ST and C4ST. C4ST transferred sulfate to position 4 of GalNAc residue adjacent to the reducing side of GlcA, while C6ST could sulfate GalNAc residue located in the IdoA rich region.
(5) We cloned mouse GalNAc4ST-1 and GalNAc4ST-2, and investigated their expression pattern, substrate specificity, and chromosomal localization. Unlike human counterpart, mouse GalNAc4ST-1 was scarcely expressed in the pituitary, but expressed strongly in the kidney, suggesting that mouse GalNAc4ST-1 is involved in the sulfation of glycoproteins in the kidney.
(6) We found that GalNAc4S-6ST is expressed in the bone marrow-derived mast cell (BMMC). The mouse GalNAc4S-6ST cloned from the BMMC could synthesize CS-E in vitro, indicating that GalNAc4S-6ST is involved in the synthesis of CS-E contained in the granule.
|
