Kozarsky K F, Call S M, Dower S K, Krieger M
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Mol Cell Biol. 1988 Aug;8(8):3357-63. doi: 10.1128/mcb.8.8.3357-3363.1988.
The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.
利用一组在蛋白质O糖基化方面存在可逆缺陷的突变型中国仓鼠卵巢（CHO）细胞系，研究了白细胞介素-2（IL-2）受体的合成及细胞内分选过程。在正常培养条件下，突变型ldlD细胞无法将N-乙酰半乳糖胺（GalNAc）添加到蛋白质上。GalNAc是连接到蛋白质上的粘蛋白型O-连接寡糖的第一个糖。当向培养基中添加GalNAc时，这种O-糖基化缺陷会迅速得到纠正。将p55人IL-2受体的表达载体转染到野生型CHO细胞和ldlD细胞中，并通过免疫沉淀和抗体结合试验检测受体的结构、稳定性和细胞表面表达情况。在添加了GalNAc的野生型CHO细胞和ldlD细胞中，基本上所有正常糖基化的IL-2受体成熟形式都表达在细胞表面。O-连接碳水化合物缺陷型（Od）IL-2受体（在未添加GalNAc的ldlD细胞中）的稳定性正常；然而，Od受体的分选错误导致细胞表面表达极少。成熟的Od IL-2受体对唾液酸酶的敏感性和对内切糖苷酶H的抗性表明，Od受体的分选错误发生在反式高尔基体或其之后。将Od IL-2受体的异常分选与低密度脂蛋白受体、衰变加速因子和爱泼斯坦-巴尔病毒主要抗原包膜蛋白的表面表达及稳定性对O-糖基化的依赖性进行了比较。
