Biosynthesis and biological roles of specific sulfation pattern of chondroitin sulfate

1.我们产生GalNAc 4S-6ST KO小鼠。GalNAc 4S-65 T-/-小鼠各组织CS-E合成活性完全丧失。GalNAc 4S-6ST-/-小鼠骨髓来源的肥大细胞不能合成CS-E，类胰蛋白酶活性降低.我们从大鼠软骨肉瘤细胞系中建立了稳定表达GalNAc 4S-6ST的克隆。该克隆合成并分泌含有CS-E的蛋白聚糖。克隆产生的CS链长远短于亲本细胞产生的CS链长，表明GalNAc 4S-6ST形成的GalNAc（4，6-SO 4）残基可能抑制了CS链的延长.我们建立了一种新的合成非还原性末端序列GalNAc的方法（4，6-SO_4）-GlcA（2SO_4）-GalNAc（6SO_4）的2 OST和GalNAc（4S-6ST）。4.通过定点突变的方法合成了C6 ST-1缺失N-糖基化位点的突变体，从鱿鱼软骨中克隆了鱿鱼GalNAc 4S-63 T的cDNA，并制备了重组酶。由重组GalNAc 4S-6ST合成的CS-E以抗凝血酶III非依赖性方式抑制凝血酶

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