Allende M L, Li J, Darling D S, Worth C A, Young W W
Department of Molecular, Cellular, and Craniofacial Biology, Schools of Dentistry and Medicine and James G. Brown Cancer Center, University of Louisville, Louisville, KY 40292, USA.
Glycobiology. 2000 Oct;10(10):1025-32. doi: 10.1093/glycob/10.10.1025.
Ganglioside GM2 synthase and other enzymes required for complex ganglioside synthesis were localized recently to the trans Golgi network (TGN). However, there are conflicting reports as to the location of GM3 synthase; originally this enzyme was detected in the early Golgi of rat liver but a recent report localized it to the late Golgi. We have used chimeric forms of ganglioside GM2 synthase to determine if the location of lactosylceramide (LacCer) to GM3 conversion in Chinese hamster ovary (CHO) cells was the early or late Golgi. Our approach tested whether GM3 could be utilized as a substrate by GM2 synthase chimeras which were targeted to compartments earlier than the trans Golgi, i.e., GM3 produced in the cis Golgi should be utilized by GM2 synthase located anywhere in the Golgi whereas GM3 produced in the trans Golgi should only be used by GM2 synthase located in the trans Golgi or TGN. Comparison of cell lines stably expressing these chimeras revealed that the in vivo functional activity of GM2 synthase decreased progressively as the enzyme was targeted to earlier compartments; specifically, the percentage of GM3 converted to GM2 was 83-86% for wild type enzyme, 70% for the medial Golgi targeted enzyme, 13% for the ER and cis Golgi targeted enzyme, and only 1.7% for the ER targeted enzyme. Thus, these data are consistent with a late Golgi location for LacCer to GM3 conversion in these cells.
神经节苷脂GM2合酶以及复杂神经节苷脂合成所需的其他酶最近定位于反式高尔基体网络（TGN）。然而，关于GM3合酶的定位存在相互矛盾的报道；最初在大鼠肝脏的早期高尔基体中检测到这种酶，但最近的一份报告将其定位于晚期高尔基体。我们使用了神经节苷脂GM2合酶的嵌合形式来确定中国仓鼠卵巢（CHO）细胞中乳糖基神经酰胺（LacCer）向GM3转化的位置是早期高尔基体还是晚期高尔基体。我们的方法测试了GM3是否可以被靶向早于反式高尔基体的区室的GM2合酶嵌合体用作底物，即顺式高尔基体中产生的GM3应该被高尔基体中任何位置的GM2合酶利用，而反式高尔基体中产生的GM3应该只被位于反式高尔基体或TGN中的GM2合酶使用。对稳定表达这些嵌合体的细胞系的比较表明，随着酶被靶向更早的区室，GM2合酶的体内功能活性逐渐降低；具体而言，野生型酶将GM3转化为GM 的百分比为83 - 86%，靶向中间高尔基体的酶为70%，靶向内质网和顺式高尔基体的酶为13%，而靶向内质网的酶仅为1.7%。因此，这些数据与这些细胞中LacCer向GM3转化发生在晚期高尔基体的位置一致。
