Uhlin-Hansen L, Kusche-Gullberg M, Berg E, Eriksson I, Kjellén L
Department of Biochemistry, Institute of Medical Biology, University of Tromso, 9037 Tromso, Norway.
J Biol Chem. 1997 Feb 7;272(6):3200-6. doi: 10.1074/jbc.272.6.3200.
In order to study the subcellular localization and organization of the enzymes involved in the glycosylation of the hybrid proteoglycan serglycin, mouse mastocytoma cells were metabolically labeled with [35S]sulfate or [3H]glucosamine in the absence or presence of brefeldin A. This drug is known to induce a disassembly of the proximal part of the Golgi complex, resulting in a redistribution of cis-, medial-, and trans-Golgi resident enzymes back to the endoplasmic reticulum, and to block the anterograde transport of proteins to the trans-Golgi network. Although the total incorporation of [3H]glucosamine into glycosaminoglycan chains was reduced to about 25% in brefeldin A-treated cells compared to control cells, both control cells and cells treated with brefeldin A synthesized heparin as well as chondroitin sulfate chains. Therefore, enzymes involved in the biosynthesis of both types of glycosaminoglycan chains seem to be present proximal to the trans-Golgi network in these cells. Chondroitin sulfate and heparin synthesized in cells exposed to brefeldin A were undersulfated, as demonstrated by ion-exchange chromatography, compositional analyses of disaccharides, as well as by a lower [35S]sulfate/[3H]glucosamine ratio compared to controls. In heparin biosynthesis, both N- and O-sulfation reactions were impaired, with a larger relative decrease in 2-O-sulfation than in 6-O-sulfation. Despite undersulfation, the heparin chains synthesized in the presence of brefeldin A were larger (30 kDa) than the heparin synthesized by control cells (20 kDa). The reduced [3H]glucosamine incorporation in brefeldin A-treated cells was partly due to decreased number of glycosaminoglycan chains synthesized, but also to the biosynthesis of chondroitin sulfate chains of smaller molecular size (8 versus 15 kDa in control cells). Brefeldin A had no effect on the glycosaminoglycan synthesis when used in a cell-free, microsomal fraction, indicating that brefeldin A does not interfere directly with the enzymes involved in the biosynthesis of glycosaminoglycans.
为了研究参与杂合蛋白聚糖丝甘蛋白聚糖糖基化的酶的亚细胞定位和组织，在有无布雷菲德菌素A的情况下，用[35S]硫酸盐或[3H]葡糖胺对小鼠肥大细胞瘤细胞进行代谢标记。已知这种药物会诱导高尔基体复合体近端部分的解体，导致顺面、中间和反面高尔基体驻留酶重新分布回内质网，并阻断蛋白质向反面高尔基体网络的顺向运输。尽管与对照细胞相比，用布雷菲德菌素A处理的细胞中[3H]葡糖胺掺入糖胺聚糖链的总量减少到约25%，但对照细胞和用布雷菲德菌素A处理的细胞都合成了肝素以及硫酸软骨素链。因此，在这些细胞中，参与这两种糖胺聚糖链生物合成的酶似乎存在于反面高尔基体网络的近端。通过离子交换色谱、二糖组成分析以及与对照相比更低的[35S]硫酸盐/[3H]葡糖胺比值证明，在暴露于布雷菲德菌素A的细胞中合成的硫酸软骨素和肝素硫酸化不足。在肝素生物合成中，N-硫酸化和O-硫酸化反应均受损，2-O-硫酸化的相对减少幅度大于6-O-硫酸化。尽管硫酸化不足，但在布雷菲德菌素A存在下合成的肝素链（30 kDa）比对照细胞合成的肝素（20 kDa）更大。在布雷菲德菌素A处理的细胞中[3H]葡糖胺掺入减少部分是由于合成的糖胺聚糖链数量减少，但也归因于分子尺寸较小的硫酸软骨素链的生物合成（对照细胞中为8 kDa，而此处为15 kDa）。当在无细胞的微粒体组分中使用时，布雷菲德菌素A对糖胺聚糖合成没有影响，这表明布雷菲德菌素A不会直接干扰参与糖胺聚糖生物合成的酶。
