Oligosaccharide Analysis of the Low Density Lipoprotein Receptors in Golgi Mutants

我们已经分离，通过独立选择，两种不同的体细胞突变，显示改变LDL受体的表达。从中国仓鼠V79细胞系中分离到致密素耐药突变体MF-2，从CHO细胞系分离到莫能菌素耐药突变体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。这些突变体中产生的成熟LDL受体似乎比亲本细胞小5000 Mr，并且突变特异性地改变了o链糖链，而不是n链糖链。这两个突变体属于同一个互补组。与乔治亚大学的R. Merkle和R. Cummings合作，进一步详细表征了int突变体的低密度脂蛋白受体中的Ser/ r-连接低聚糖。亲本和Mon^r-31细胞受体中的丝氨酸/丝氨酸连接寡糖是单和双唾液化的，具有共同的核心结构Gal-GalNAc。然而，来自Mon^r-31细胞的受体比来自亲代CHO细胞的受体含有约三分之一的丝氨酸/丝氨酸连接的低聚糖。在CHO中缺乏簇状丝氨酸/丝氨酸连接寡糖的人突变受体的成熟形式的表观M_r为120000，而在Mon^r-31中，其M_r为110000。唾液酸酶处理的人突变受体在more ^r-31细胞中的表观相对分子质量与同时用唾液酸酶和o -聚糖酶处理的CHO细胞中的受体相当。这些结果表明，由Mon^r-31细胞产生的LDL受体在聚集域含有丝氨酸/ thrr -连接的低聚糖，但在受体的非聚集LDL结合域缺少丝氨酸/ thrr -连接的低聚糖。低密度脂蛋白结合区域的o -糖基化可能对低密度脂蛋白受体的功能至关重要。

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.