Locker J K, Griffiths G, Horzinek M C, Rottier P J
European Molecular Biology Laboratory, Heidelberg, Germany.
J Biol Chem. 1992 Jul 15;267(20):14094-101. doi: 10.1016/S0021-9258(19)49683-X.
It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain A59 (MHV-A59) contains only O-linked oligosaccharides and localizes to the Golgi region when expressed independently. A detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.
先前的研究表明，小鼠肝炎病毒A59株（MHV - A59）的M（E1）糖蛋白仅含有O - 连接寡糖，当独立表达时定位于高尔基体区域。对M蛋白上O - 连接糖的添加进行了详细的脉冲追踪分析；在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上，可以区分出三种不同的电泳形式，它们分别对应于依次获得N - 乙酰半乳糖胺（GalNAc）、半乳糖（Gal）和唾液酸（SA）。还可以检测到第四种和第五种形式，但我们无法鉴定它们。用布雷菲德菌素A处理后，M蛋白仍能获得GalNAc、Gal和SA，但第四种和第五种形式不存在，这表明这些修饰发生在反式高尔基体网络（TGN）中。相比之下，在布雷菲德菌素A存在的情况下，含有N - 连接寡糖的水泡性口炎病毒（VSV）的G蛋白获得了Gal和岩藻糖，但没有获得SA。这些结果与早期发表的数据一致，表明TGN近端的高尔基体区室（而非TGN本身）会重新定位到内质网/中间区室。更重要的是，我们的数据表明，虽然N - 连接糖上SA的添加发生在TGN中，但O - 连接糖上SA的获得以及N - 连接寡糖上岩藻糖的添加必定发生在TGN近端的高尔基体区室中。此外，M蛋白的糖基化表明它被转运到反式高尔基体和TGN。这通过电子显微镜免疫细胞化学得到了证实，显示该蛋白靶向高尔基体反侧的扁平囊，并且至少部分地与TGN的标志物TGN 38以及对唾液酸特异的凝集素共定位。
