Esko J D, Stewart T E, Taylor W H
Proc Natl Acad Sci U S A. 1985 May;82(10):3197-201. doi: 10.1073/pnas.82.10.3197.
We have obtained Chinese hamster ovary cell mutants defective in the biosynthesis of glycosaminoglycans by screening replicate colonies immobilized on polyester cloth. Depending upon the strain, the mutants accumulated less 35S-labeled glycosaminoglycans per microgram of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, five strains failed to generate 3H-labeled glycosaminoglycans normally. In four of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-beta-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.
我们通过筛选固定在聚酯布上的重复菌落，获得了在糖胺聚糖生物合成方面存在缺陷的中国仓鼠卵巢细胞突变体。根据菌株的不同，与野生型相比，这些突变体每微克细胞蛋白积累的35S标记糖胺聚糖减少了6至60倍。一些突变体将[6-3H]葡萄糖胺掺入糖胺聚糖的程度与野生型相同，这表明硫酸盐添加发生了特异性改变。相比之下，有五个菌株不能正常产生3H标记的糖胺聚糖。在其中四个菌株中，糖胺聚糖组装的起始发生了特异性改变，因为添加对硝基苯基-β-木糖苷可使硫酸化恢复正常。对这些突变体提取物中的木糖基转移酶进行酶活性测定发现，其中一个菌株S745的酶活性比野生型低15倍。这个突变体提供了遗传学证据，证明体外测定的木糖基转移酶在体内负责硫酸软骨素和硫酸乙酰肝素生物合成的起始。
