Biosynthesis and biological roles of specific sulfation pattern of chondroitin sulfate

1. 我们生成GalNAc4S-6ST KO小鼠。GalNAc4S-65T-/-小鼠各组织中CS-E合成活性完全丧失。GalNAc4S-6ST-/-小鼠骨髓源肥大细胞不能合成CS-E，并表现出胰蛋白酶活性降低。我们从大鼠软骨肉瘤细胞系中建立了表达GalNAc4S-6ST的稳定克隆。该克隆合成并分泌含有CS-E的蛋白多糖。克隆产生的CS链长比亲本细胞产生的CS短得多，表明GalNAc4S-6ST形成GalNAc （4,6- so4）残基可能抑制了链长。以GalNAc(4SO_4)-GlcA-GalNAc（6SO_4）为原料，以2OST和GalNAc4S-6ST为原料，建立了独特的不还原末端序列GalNAc(4,6- so_4)-GlcA(2SO_4)-GalNAc（6SO_4）的合成方法。通过位点定向诱变，我们合成了多种缺乏n -糖基化位点的C6ST-1突变体，结果表明，附着在C6ST-1上的n -聚糖参与了c6st - i - 5活性酶的产生、细胞内定位和底物特异性。从鱿鱼软骨中克隆鱿鱼GalNAc4S-63T cDNA，制备重组酶。重组GalNAc4S-6ST合成的CS-E以不依赖抗凝血酶iii的方式抑制凝血酶

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