Esko J D, Elgavish A, Prasthofer T, Taylor W H, Weinke J L
J Biol Chem. 1986 Nov 25;261(33):15725-33.
We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans. Thirty-five mutants incorporated [6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake. Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparan and chondroitin chains. Incubation of the mutant with [35S]cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing [35S]cysteine, and isotope dilution analysis indicated that about 15 microM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 microM sodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not proliferate in medium containing 5 microM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.
我们分离出了59个中国仓鼠卵巢细胞突变体，这些突变体在将35SO4掺入糖胺聚糖方面存在缺陷。35个突变体将[6-3H]葡萄糖胺正常掺入糖胺聚糖，这表明它们在硫酸盐掺入方面存在特异性缺陷。细胞杂交研究表明，这35个突变体定义了一个独特的互补群。用35SO4对其中一个突变体进行脉冲标记显示，它在硫酸盐摄取所需的一种可饱和的、对4-乙酰氨基-4-异硫氰酸基芪-2,2'-二磺酸敏感的转运系统中存在缺陷。尽管35SO4掺入量大幅减少，但该突变体仍合成了硫酸化的硫酸乙酰肝素和软骨素链。用[35S]半胱氨酸孵育该突变体导致形成35SO4，随后其被掺入糖胺聚糖中。当野生型细胞在含有[35S]半胱氨酸的无硫酸盐生长培养基中孵育时，也获得了类似结果，同位素稀释分析表明约15 microM的硫酸盐来自半胱氨酸分解代谢。我们还发现，硫酸盐转运缺陷使该突变体对5 microM铬酸钠具有抗性，而野生型细胞在这些条件下不分裂。然而，当在野生型细胞存在的情况下生长时，该突变体在含有5 microM铬酸盐的培养基中也不增殖，这表明铬酸盐是通过细胞间接触进行转运的。由于。由于将硫酸盐转运缺陷突变体与木糖基转移酶或半乳糖基转移酶I缺陷的突变体共培养可部分恢复35SO4掺入糖胺聚糖，因此细胞间的硫酸盐转运也会发生。因此，用于糖胺聚糖合成的硫酸盐可用性取决于硫酸盐摄取、含硫氨基酸的周转以及细胞间的硫酸盐转运。
