Lyon M, Steward W P, Hampson I N, Gallagher J T
Biochem J. 1987 Mar 1;242(2):493-8. doi: 10.1042/bj2420493.
The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans.
研究了硫酸乙酰肝素链中N-硫酸基团的分布情况。从成人皮肤成纤维细胞中提取的可被去污剂提取的硫酸乙酰肝素蛋白聚糖，用[³H]葡萄糖胺和[³⁵S]硫酸盐进行放射性标记后，与溴化氰活化的琼脂糖4B偶联。在用亚硝酸对硫酸乙酰肝素进行部分解聚后，通过碱处理去除剩余的与琼脂糖结合的片段。这些大小各异但均含有完整还原木糖残基的片段在Sephacryl S-300上进行分级分离，并分析³H和³⁵S放射性标记的分布。观察到链的还原末端硫酸化程度降低。在对与琼脂糖结合的蛋白聚糖进行完全亚硝酸水解后，对N-硫酸化与还原末端的接近程度进行分析，结果显示在与蛋白质连接序列直接相邻的位置存在一个延伸的N-乙酰化序列。通过凝胶过滤估计该N-乙酰化结构域的大小约为八个二糖单位。该结构域似乎高度保守，几乎存在于源自该蛋白聚糖的所有链中，这意味着在硫酸乙酰肝素的聚合后修饰过程中存在一种能够产生这种非随机序列的机制。与肝素中的相应情况进行比较表明，不同的机制调节这些化学相关的糖胺聚糖在蛋白质连接区域附近的聚合物N-硫酸化。
