Oligosaccharide Analysis of the Low Density Lipoprotein Receptors in Golgi Mutants

通过独立选择，我们分离到了两个不同的体细胞突变体，它们的低密度脂蛋白受体的表达发生了变化。从中国仓鼠V79细胞系中分离到一个紧缩素抗性突变体MF-2，从中国仓鼠细胞系CHO中分离到一个莫能菌素抗性突变体(Mon^r-31)。这两个突变体对低密度脂蛋白的反应降低，胆固醇代谢异常。这些突变体产生的成熟低密度脂蛋白受体似乎比亲本细胞的小5000 mR，而且突变专门改变了O-连接的糖链，而不是N-连接的糖链。这两个突变体属于同一互补INT群。与乔治亚大学的R.Merkle和R.Cummings合作，进一步详细描述了INT突变体的低密度脂蛋白受体中Ser/Thr连接的寡糖的特征。亲本细胞和Mon^r-31细胞受体中的Ser/Thr连接的寡糖都是单唾液酸化和双唾液酸化，具有共同的核心结构Gal-GalNAc。然而，来自Mon^r-31细胞的受体含有的丝氨酸/苏氨酸连接的寡糖比来自亲本CHO细胞的受体少约三分之一。缺乏簇状丝氨酸/苏氨酸连接的寡糖的人突变受体成熟型在CHO中的表观MR为120000，而在Mon^r-31中的表观MR为110000。唾液酸酶处理的人突变受体在MOR^R-31细胞中的表观相对分子质量与唾液酸酶和O-葡聚糖酶处理的CHO细胞中的受体的表观相对分子质量相当。这些结果表明，Mon^r-31细胞产生的低密度脂蛋白受体在聚集区含有丝氨酸/苏氨酸连接的寡糖，但在未聚集的低密度脂蛋白结合区缺少丝氨酸/苏氨酸连接的寡糖。低密度脂蛋白结合区的O-糖基化可能是低密度脂蛋白受体功能的关键。

We have isolated, by independent selection, two different somatic cell mutants that show altered expression of LDL receptor. A compactin-resistant mutant, MF-2, was isolated from the Chinese hamster V79 cell line and a monensin-resistant (Mon^r-31) mutant from CHO. These two mutants showed a decreased response to LDL as well as aberrant cholesterol metabolism. The mature LDL receptors produced in these mutants were seemingly 5000 Mr smaller than that of the parental cells, and the mutation specifically alters O-linked sugar chains, not N-linked sugar chains. These two mutants fall into the same complementation int group. Further detailed characterization of the Ser/Thr-linked oligosaccharides in the LDL receptor of the int mutant was done in collaboration with R. Merkle and R. Cummings at the University of Georgia. The Ser/Thr-linked oligosaccharides in the receptors from both the parental and Mon^r-31 cells are mono- and di-sialylated species having the common core structure Gal-GalNAc. The receptor from Mon^r-31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. The mature form of the human mutant receptor lacking the clustered Ser/Thr-linked oligosaccharides in CHO has an apparent M_r of 120000 while that in Mon^r-31 has a M_r of 110000. The apparent relative molecular mass of the sialidase-treated human mutant receptor in Mor^r-31 cells is equivalent to that of the receptor in CHO cells that is treated with both sialidase and O-glycanase. These results demonstrate that the LDL receptor produced by the Mon^r-31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain, but is missing the Ser/Thr-linked oligosaccharides in the unclustered LDL-binding domains of the receptor. The O-glycosylation at the LDL-binding domain may be critical for LDL receptor function.