Kreisel W, Hanski C, Tran-Thi T A, Katz N, Decker K, Reutter W, Gerok W
Klinikum Albert-Ludwigs-Universität, Freiburg, Federal Republic of Germany.
J Biol Chem. 1988 Aug 25;263(24):11736-42.
The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.
本文展示了大鼠肝细胞质膜糖蛋白二肽基肽酶IV（DPP IV）的末端去糖基化和再糖基化过程。在脉冲追踪实验中，使用培养的肝细胞，以[3H]L-岩藻糖和[14C]N-乙酰-D-甘露糖胺作为末端碳水化合物的标记物，[3H]D-甘露糖作为核心糖的标记物，[35S]L-甲硫氨酸用于标记蛋白质骨架。膜DPP IV用一种多克隆抗体进行免疫沉淀，该抗体在4℃时选择性地结合细胞表面糖蛋白。肝细胞质膜DPP IV的最大标记时间分别为：[3H]L-岩藻糖6 - 9分钟，[3H]D-甘露糖20分钟，[35S]L-甲硫氨酸25分钟。当抗体在4℃与细胞表面DPP IV结合时，尽管存在持续的代谢和膜转运过程，但在复温至37℃后免疫复合物保持稳定超过1小时。这通过在37℃用[35S]L-甲硫氨酸进行脉冲标记，然后冷却至4℃，并加入抗质膜DPP IV抗体得以证明。在复温过程中，复合物中的放射性保持恒定。在使用[3H]L-岩藻糖的类似实验中，免疫复合物中的放射性迅速下降，表明质膜糖蛋白发生了去岩藻糖基化。使用相同的实验设计但用[3H]D-甘露糖，免疫复合物中的放射性保持恒定，表明核心糖D-甘露糖未从膜糖蛋白上裂解下来。末端再糖基化（重新岩藻糖基化和重新唾液酸化）如下所示。肝细胞在补充了衣霉素的培养基中于37℃培养，以阻断N-糖苷键结合的碳水化合物链的从头合成。在4℃时，抗DPP IV抗体仅结合细胞表面糖蛋白。在37℃的复温期间，[3H]L-岩藻糖和[14C]N-乙酰-D-甘露糖胺的放射性被掺入免疫复合物中。这表明原本存在于细胞表面的糖蛋白发生了岩藻糖基化和唾液酸化。文中讨论了在膜循环过程中质膜糖蛋白末端去糖基化和再糖基化可能发生的机制。
