USE OF A MUTANT-CELL LINE TO STUDY THE KINETICS AND FUNCTION OF O-LINKED GLYCOSYLATION OF LOW-DENSITY LIPOPROTEIN RECEPTORS
USE OF A MUTANT-CELL LINE TO STUDY THE KINETICS AND FUNCTION OF O-LINKED GLYCOSYLATION OF LOW-DENSITY LIPOPROTEIN RECEPTORS
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DOI：
10.1073/pnas.85.12.4335
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发表时间：
1988-06-01
影响因子：
11.1
通讯作者：
KRIEGER, M
中科院分区：
文献类型：
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作者：
KOZARSKY, K;KINGSLEY, D;KRIEGER, M
利用中国仓鼠卵巢（CHO）细胞突变株的蛋白质O-糖基化缺陷，研究了低密度脂蛋白（LDL）受体O-糖基化的动力学和功能。突变株，基因型LDLD，不能在正常培养条件下合成UDP-N-乙酰半乳糖胺，因此，不能添加粘蛋白型O-连接的寡糖蛋白质。当向培养基中加入N-乙酰半乳糖胺时，LDLD细胞中的UDP-N-乙酰半乳糖胺池可以迅速填充，从而恢复O-连接碳水化合物的正常合成。脉冲追踪代谢标记实验表明：（i）LDL受体O-糖基化的第一步可以在细胞后发生;（ii）O-连接糖缺陷型LDL受体到达细胞表面后，它们不受随后O-连接糖添加的影响，表明它们不返回O-糖基化发生的区室;（iii）LDL受体自身上的O-连接的碳水化合物链是正常稳定性和功能所必需的;和（iv）O-连接的糖缺陷型LDL受体的不稳定性是由于蛋白水解裂解和受体的NH 2-末端胞外结构域大量释放到介质中。似乎LDL受体和几种其他细胞表面糖蛋白的O-糖基化通过阻止这些蛋白质的胞外结构域的蛋白水解裂解而允许稳定的细胞表面表达。
A rapidly reverible defect in protein O-glycosylation exhibited by a line of mutant Chinese hamster ovary (CHO) cells was used to study the kinetics and function of O-glycosylation of the low density lipoprotein (LDL) receptor. The mutant line, genotype LDLD, cannot synthesize UDP-N-acetlygalactosamine under normal culture conditions and, therefore, cannot add mucin-type O-linked oligosaccharides to proteins. The UDP-N-acetylalactosamine pools in LDLD cells can be filled rapidly when N-acetylgalactosamine is added to the culture medium, thus restoring normal synthesis of O-linked carbohydrates. Pulse-chase metabolic labeling experiments were used to show that (i) the first step in the O-glycosylation of LDL receptors can occur posttranslationally; (ii) after O-linked sugar-deficient LDL receptors reach the cell surface, they are not subject to subsequent O-linked sugar addition, suggesting that they do not return to compartments in which O-glycosylation takes place; (iii) O-linked carbohydrate chains on the LDL receptor itself are required for normal stability and function; and (iv) the instability of the O-linked sugar-deficient LDL receptor is due to proteolytic cleavage and the release into the medium of the bulk ofthe NH2-terminal extracellular domain of the receptor. It appears that O-glycosylation of the LDL receptor and several other cell surface glycoproteins permits stable cell-surface expression by preventing proteolytic cleavage of the extracellular domains of these proteins.