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

(1)建立了一种能在体外合成CS-E的酶促方法。(2)我们在硫酸软骨素中发现了一个独特的非还原末端结构GalNAc(4-SO_4)-GlCa(2SO_4)-GalNAc(6SO_4)，该结构被GalNAc4S-6st转化为一个高度硫酸盐化的序列(GalNAc(4，6-SO_4)-Glca(2SO_4)-GalNAc(6SO_4))。在培养的鸡软骨细胞合成的硫酸软骨素中发现了高度硫酸盐化的非还原序列，表明这种结构似乎普遍存在于软骨硫酸软骨素中。(3)连接在C4ST上的N-连接寡糖被发现是酶活性所必需的。C4ST C末端低聚糖的缺失不仅导致酶活性的急剧下降，而且导致糖基化模式的改变。(4)重组C6ST和C4ST能有效地对脱硫化皮肤素硫酸酯进行硫酸盐化。C4ST将硫酸盐转移到GalNAc残基靠近Glca还原侧的第4位，而C6ST可以将GalNAc残基转移到IoA富集区。(5)克隆了小鼠GalNAc4ST-1和GalNAc4ST-2，并对其表达模式、底物特异性和染色体定位进行了研究。与人类不同，小鼠GalNAc4ST-1在脑垂体中几乎不表达，但在肾脏中表达较强，提示小鼠GalNAc4ST-1参与了肾脏糖蛋白的硫酸化。(6)我们发现GalNAc4S-6ST在骨髓源性肥大细胞(BMMC)中有表达。从BMMC克隆的小鼠GalNAc4S-6st在体外可以合成CS-E，表明GalNAc4S-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.