Smith M E
Department of Neurology, Veterans Administration Medical Center, Palo Alto, CA 94304.
J Neurochem. 1991 Aug;57(2):655-64. doi: 10.1111/j.1471-4159.1991.tb03797.x.
Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.
使用四种寡糖加工抑制剂来研究它们对周围神经髓鞘糖蛋白通过分泌途径转运的影响，并进一步深入了解P0和19-kDa糖蛋白寡糖链的结构。将几种不同的寡糖加工抑制剂与幼鼠（21 - 24日龄）的切碎外周神经一起孵育，在聚丙烯酰胺凝胶上分离匀浆和髓鞘蛋白后，测量P0和19-kDa糖蛋白中14C-氨基酸以及[3H]岩藻糖或[3H]甘露糖的摄取量。发现[3H]甘露糖作为寡糖前体不如[3H]岩藻糖合适，因为用作能量来源的葡萄糖会显著抑制[3H]甘露糖的摄取。然而，用丙酮酸替代能量来源会导致糖基化不完全、氨基酸摄取不良以及寡糖链截短。内切糖苷酶H切割了约50%用[3H]岩藻糖和14C-氨基酸标记的P0。内切糖苷酶H切割产生的较低分子量蛋白质在蛋白质上所含的[3H]岩藻糖标记约为一半，而另一半保留在未降解P0的寡糖链上，这表明至少一半的P0具有杂合结构。脱氧野尻霉素、脱氧甘露野尻霉素和栗精胺抑制了[DH]岩藻糖掺入P0和19-kDa糖蛋白的寡糖链中，这与它们在添加岩藻糖之前阻断高甘露糖结构修剪各个阶段的作用预测一致。在这些抑制剂存在下合成的P0比正常蛋白质更易被内切糖苷酶H切割，这表明随着正常加工的停滞，对该酶的敏感性增加。19-kDa糖蛋白也得到了类似的结果。在这些抑制剂存在下形成的加工不完全 的P0和19-kDa糖蛋白似乎都能正常转运到髓鞘中。
