Study on the function and control of sulfotransferases involving the synthesis of divergent structure of chondroitin sulfate

(1)我们开发了一种能够在体外合成CS-E的酶促方法。酶促合成的CS-E与中期因子强烈结合。(2)我们发现硫酸软骨素中存在一种独特的非还原性末端结构GalNAc（4-SO_4）-GlcA（2SO_4）-GalNAc（6SO_4），并发现这种结构被GalNAc 4S-6ST转化为高度硫酸化的序列（GalNAc（4，6-SO_4）-GlcA（2SO_4）-GalNAc（6SO_4）。高度硫酸化的非还原序列被发现在软骨素硫酸合成的培养的鸡软骨细胞，表明这种结构似乎是普遍存在于软骨硫酸软骨素。(3)N-连接的寡糖连接到C4 ST被认为是所需的酶的活性。C4 ST的C-末端区域的寡糖的缺失不仅导致活性的急剧下降，而且在糖基化模式的改变。(4)通过重组C6 ST和C4 ST有效地硫酸化脱硫硫酸皮肤素。C4 ST将硫酸根转移到GlcA还原侧附近的GalNAc残基的4位，而C6 ST可将位于IdoA富集区的GalNAc残基硫酸化。(5)我们克隆了小鼠GalNAc 4ST-1和GalNAc 4ST-2，并研究了它们的表达模式、底物特异性和染色体定位。与人类对应物不同，小鼠GalNAc 4ST-1在垂体中几乎不表达，但在肾脏中强烈表达，表明小鼠GalNAc 4ST-1参与肾脏中糖蛋白的硫酸化。(6)我们发现GalNAc 4S-6ST在骨髓源性肥大细胞（BMMC）中表达。从BMMC中克隆的小鼠GalNAc 4S-6ST在体外可合成CS-E，表明GalNAc 4S-6ST参与了颗粒中CS-E的合成。

(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.