Oligosaccharide Analysis of the Low Density Lipoprotein Receptors in Golgi Mutants

高尔基体突变体低密度脂蛋白受体的寡糖分析

• 批准号: 63044114
• 负责人: KUWANO Michihiko
• 金额: $ 3.14万
• 依托单位: Oita Medical School, Faculty of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63044114/
• 关键词: low density lipoprotein (LDL); LDL-receptor; familiar hypercholesterolemia; receptor mutation; somatic cell mutants; Golgi apparatus; O-linked sugar chains; cholesterol metabolism

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.

我们已经分离出，通过独立的选择，两种不同的体细胞突变体，表现出改变LDL受体的表达。从中国仓鼠V79细胞系中分离出一种抗致密化蛋白突变体MF-2，并从CHO中分离出一种抗莫能菌素突变体（Mon^r-31）。这两个突变体表现出对LDL的反应降低以及胆固醇代谢异常。在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群。与R. Merkle和R.卡明斯在格鲁吉亚大学。来自亲本和Mon^r-31细胞的受体中的Ser/Thr连接的寡糖是具有共同核心结构Gal-GalNAc的单唾液酸化和二唾液酸化物质。然而，来自Mon^r-31细胞的受体含有的Ser/Thr连接的寡糖比来自亲本CHO细胞的受体少约三分之一。缺失成簇的Ser/Thr连接的寡糖的人突变受体的成熟形式在CHO中具有120000的表观M_r，而在Mon^r-31中具有110000的M_r。莫尔^r-31细胞中唾液酸酶处理的人突变型受体的表观相对分子质量与用唾液酸酶和O-聚糖酶处理的CHO细胞中受体的表观相对分子质量相当。这些结果表明，由Mon^r-31细胞产生的LDL受体在成簇结构域中含有Ser/Thr连接的寡糖，但在受体的非成簇LDL结合结构域中缺少Ser/Thr连接的寡糖。LDL结合结构域的O-糖基化可能对LDL受体功能至关重要。