Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.
Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.
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Mr 46,000 甘露糖 6-磷酸受体的糖基化。
DOI：
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发表时间：
1991
影响因子：
4.8
通讯作者：
R. Pohlmann
中科院分区：
文献类型：
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作者：
M. Wendland;A. Waheed;B. Schmidt;A. Hille;G. Nagel;K. von Figura;R. Pohlmann
利用定点诱变技术，将Mr 46000甘露糖6-磷酸受体（MPR 46）的n -糖基化位点鉴定为天冬酰胺残基57、83、107和113。两个外部天冬酰胺携带高甘露糖型，两个内部天冬酰胺携带复合体型寡糖。分析了糖基化突变体的稳定性、结合活性和亚细胞分布。用苏氨酸代替天冬酰胺57、83或107只会降低受体的稳定性。用苏氨酸替代天冬酰胺113降低了其稳定性和结合活性。三个或全部四个n -糖基化位点的缺失导致突变受体在内质网状结构中积累。在tunicamycin存在下合成的非糖基化MPR 46，因此保留了天冬酰胺残基，具有正常的稳定性和高亲和力结合。因此，受体突变体的稳定性和结合活性的降低是由于天冬酰胺残基的交换，而不是由于n链低聚糖的损失。然而，非糖基化受体在溶解后表现出较低的构象稳定性，因为冷冻和解冻的单一循环将结合活性降低到对照的三分之一。同时，受体失去了它的四级结构。从这些结果可以得出结论，受体的n -糖基化对于高亲和力构象的稳定性是必需的，而不是结合本身或细胞内稳定性所必需的。
Using site-directed mutagenesis the N-glycosylation sites of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) were identified as asparagine residues 57, 83, 107, and 113. The two outer asparagines carry high mannose-type and the two inner asparagines carry complex-type oligosaccharides. The glycosylation mutants were analyzed for stability, binding activity, and subcellular distribution. Replacing asparagine 57, 83, or 107 by threonine decreased only the stability of the receptor. Replacing asparagine 113 by threonine decreased the stability and binding activity. Deletion of three or all four N-glycosylation sites led in addition to an accumulation of the mutant receptors in endoplasmic reticulum-like structures. Nonglycosylated MPR 46 synthesized in the presence of tunicamycin, thus preserving the asparagine residues, had a normal stability and high affinity binding. The decreased stability and binding activity of the receptor mutants is therefore due to the exchange of asparagine residues rather than to the loss of N-linked oligosaccharides. The nonglycosylated receptor, however, displayed a decreased conformational stability after solubilization as a single cycle of freezing and thawing reduced the binding activity to one-third of the control. Simultaneously, the receptor lost its quaternary structure. It is concluded from these results that the N-glycosylation of the receptor is required for the stability of a high affinity conformation, but not for the binding itself or the intracellular stability.