Arakaki R F, Hedo J A, Collier E, Gorden P
J Biol Chem. 1987 Aug 25;262(24):11886-92.
The insulin proreceptor is a 190-kDa glycoprotein that is processed to mature alpha (135-kDa) and beta (95-kDa) subunits. In order to determine the role of carbohydrate chain processing in insulin receptor biogenesis, we investigated the effect of inhibiting glucose removal from core oligosaccharides of the insulin proreceptor with glucosidase inhibitors, castanospermine and 1-deoxynojirimycin. Cultured IM-9 lymphocytes treated with inhibitors had 50% reduction in surface insulin receptors as demonstrated by ligand binding, affinity cross-linking with 125I-insulin, and lactoperoxidase/Na 125I labeling studies. Degradation rates of surface labeled receptors were similar in both control and inhibitor-treated cells (t1/2 = 5 h); thus, accelerated receptor degradation could not account for this reduction. Biosynthetic labeling experiments with [3H]leucine and [3H]mannose identified an apparently higher molecular size proreceptor (approximately 205 kDa) that failed to show the characteristic decline with time as seen in the normal 190-kDa proreceptor. Along with this finding, the biosynthetic label appearing in the mature subunits was reduced in these inhibitor-treated cells. Endoglycosidase H treatment of both precursors produced identical 170-kDa bands. Carbohydrate chains released from the 205-kDa precursor by endoglycosidase H migrated in the same position as the Glc2-3Man9GlcNAc standards when separated by high performance liquid chromatography, whereas the 190-kDa proreceptor oligosaccharides migrated similar to the Man7-9GlcNAc chains. Although the mature subunits of control and inhibitor-treated cells demonstrated equal electrophoretic mobility, the endoglycosidase H-sensitive oligosaccharides of the mature subunits in treated cells also contained residues that migrated similar to the Glc2-3Man9GlcNAc standards. Thus, glucose removal from core oligosaccharides is apparently not necessary for the cleavage of the insulin proreceptor, but does delay processing of this precursor, which probably accounts for the reduction in cell-surface receptors.
胰岛素原受体是一种190 kDa的糖蛋白，可加工成成熟的α亚基（135 kDa）和β亚基（95 kDa）。为了确定碳水化合物链加工在胰岛素受体生物合成中的作用，我们研究了用葡萄糖苷酶抑制剂、蓖麻毒蛋白和1-脱氧野尻霉素抑制胰岛素原受体核心寡糖中葡萄糖去除的效果。如通过配体结合、用125I-胰岛素进行亲和交联以及乳过氧化物酶/Na 125I标记研究所示，用抑制剂处理的培养IM-9淋巴细胞表面胰岛素受体减少了50%。对照细胞和用抑制剂处理的细胞中表面标记受体的降解速率相似（t1/2 = 5小时）；因此，受体降解加速不能解释这种减少。用[3H]亮氨酸和[3H]甘露糖进行的生物合成标记实验确定了一种明显分子量更大的原受体（约205 kDa），它没有像正常的190 kDa原受体那样随时间呈现出特征性的下降。伴随着这一发现，在这些用抑制剂处理的细胞中，出现在成熟亚基中的生物合成标记减少了。用内切糖苷酶H处理这两种前体均产生相同的170 kDa条带。当通过高效液相色谱分离时，内切糖苷酶H从205 kDa前体释放的碳水化合物链迁移到与Glc2-3Man9GlcNAc标准相同的位置，而190 kDa原受体寡糖的迁移类似于Man7-9GlcNAc链。尽管对照细胞和用抑制剂处理的细胞的成熟亚基表现出相同的电泳迁移率，但处理细胞中成熟亚基对内切糖苷酶H敏感的寡糖也含有迁移类似于Glc2-3Man9GlcNAc标准的残基。因此，从核心寡糖中去除葡萄糖显然不是胰岛素原受体裂解所必需的，但确实会延迟该前体的加工，这可能是细胞表面受体减少的原因。
