Perkel V S, Liu A Y, Miura Y, Magner J A
Division of Endocrinology, Michael Reese Hospital, University of Chicago, Illinois 60616.
Endocrinology. 1988 Jul;123(1):310-8. doi: 10.1210/endo-123-1-310.
We have studied the effects of Brefeldin-A (BFA) on the processing of high mannose (Man) oligosaccharides of TSH. BFA is a drug that inhibits the intracellular translocation of newly synthesized glycoproteins and causes dilatation of the rough endoplasmic reticulum (RER) as well as mild swelling of the Golgi apparatus. Mouse pituitary thyrotropic tumor tissue was incubated with [3H]Man for a 2-h pulse, with and without a 3-h chase; BFA (5 micrograms/ml) was included during selected pulse and selected chase incubations. TSH and free alpha-subunits were obtained from detergent lysates of tissue by immunoprecipitation using specific antisera. Total glycoproteins were obtained by trichloroacetic acid precipitation. Endoglycosidase-H-released [3H]oligosaccharides were analyzed by paper chromatography. BFA inhibited carbohydrate processing of TSH, free alpha-subunits, and total glycoproteins, resulting in the accumulation of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2, especially during the chase period. Subcellular fractions enriched in RER, heavy (proximal) Golgi, and light (distal) Golgi were prepared by centrifugation in discontinuous sucrose gradients. [3H]Man-labeled oligosaccharides of TSH and total glycoproteins in the subcellular fractions were analyzed. In contrast to oligosaccharides with eight or nine Man residues found in control incubations, BFA caused the accumulation of oligosaccharides containing five to eight Man residues. These BFA-induced oligosaccharide alterations began in the RER and proximal Golgi with the 2-h pulse and extended into the distal Golgi during the chase incubations. Thus, BFA blocks the normal intracellular transport and processing of TSH, free alpha-subunits, and total glycoproteins within thyrotrophs, causing species with smaller than normal high Man oligosaccharides to appear in subcellular compartments as early as the RER. The translocation block between RER and Golgi produced by BFA may prevent the processing of Man8GlcNAc2 to Man5GlcNAc2 by Golgi (alpha,1-2)mannosidase I, yet the species retained within the RER may be subject to ongoing processing by endoplasmic reticulum (alpha,1-2)mannosidase, resulting in the accumulation of Man5-8GlcNAc2 within the RER.
我们研究了布雷菲德菌素 A（BFA）对促甲状腺激素（TSH）高甘露糖（Man）寡糖加工过程的影响。BFA 是一种能抑制新合成糖蛋白细胞内转运的药物，可导致粗面内质网（RER）扩张以及高尔基体轻度肿胀。将小鼠垂体促甲状腺肿瘤组织与[3H]Man 一起进行 2 小时脉冲孵育，有或没有 3 小时的追踪孵育；在选定的脉冲和追踪孵育期间加入 BFA（5 微克/毫升）。通过使用特异性抗血清进行免疫沉淀，从组织的去污剂裂解物中获得 TSH 和游离α亚基。通过三氯乙酸沉淀获得总糖蛋白。通过纸层析分析内切糖苷酶 H 释放的[3H]寡糖。BFA 抑制了 TSH、游离α亚基和总糖蛋白的碳水化合物加工过程，导致 Man8GlcNAc2、Man7GlcNAc2、Man6GlcNAc2 和 Man5GlcNAc2 的积累，尤其是在追踪期间。通过在不连续蔗糖梯度中离心制备富含 RER、重（近端）高尔基体和轻（远端）高尔基体的亚细胞组分。分析亚细胞组分中 TSH 和总糖蛋白的[3H]Man 标记寡糖。与对照孵育中发现的含有八个或九个 Man 残基的寡糖不同，BFA 导致含有五到八个 Man 残基的寡糖积累。这些 BFA 诱导的寡糖变化在 2 小时脉冲时开始于 RER 和近端高尔基体，并在追踪孵育期间延伸到远端高尔基体。因此，BFA 阻断了促甲状腺细胞内 TSH、游离α亚基和总糖蛋白的正常细胞内转运和加工过程，导致在亚细胞区室中最早在 RER 就出现比正常高 Man 寡糖更小的糖型。BFA 在 RER 和高尔基体之间产生的转运阻断可能会阻止高尔基体（α，1-2）甘露糖苷酶 I 将 Man8GlcNAc2 加工为 Man5GlcNAc2，然而保留在 RER 内的糖型可能会受到内质网（α，1-2）甘露糖苷酶的持续加工，导致 Man5-8GlcNAc2 在 RER 内积累。
