GANGLIOSIDE BIOSYNTHESIS IN TUMOR CELLS

神经节苷脂（唾液酸化鞘糖脂）是神经节苷脂的普遍存在的组分， 哺乳动物细胞神经节苷脂表达在许多肿瘤类型中改变， 特别是在恶性黑素瘤、神经母细胞瘤和星形细胞瘤中。 神经节苷脂也是这些肿瘤类型中的重要抗原。而 神经节苷脂在肿瘤中的表达模式现在很好， 了解，很少有人知道是什么决定了特性 不同细胞类型中的模式和以下改变的表达 转型这个项目的主要目标是了解 肿瘤细胞中神经节苷脂的表达是确定和调节的。 重点将是对各种糖基转移酶的分析 负责恶性肿瘤中神经节苷脂的生物合成 黑色素瘤和神经母细胞瘤细胞。我们之前研究过 一组细胞系中的糖基转移酶水平和活性， 确定了这些细胞中神经节苷脂生物合成的关键步骤。 GD 3合成酶（SAT-2）和GM 2\GD 2合成酶（GalNAc-T）在大肠杆菌中的表达较强， 这一过程中的重要酶。我们最近分离了一个cDNA克隆， 编码GalNAc-T，并研究了它的一些性质。这些研究将 通过（i）继续表征GalNAc-T基因及其 产品，（ii）产生这种酶的抗体，（iii）使用这些 分离酶并检查其糖基化的抗体，（iv） 尝试克隆SAT-2的cDNA，（v）纯化该酶并研究其 性质，和（vi）产生针对SAT-2的抗体。使用这些试剂，我们 将开始对控制进行更详细的分析 决定这些肿瘤的神经节苷脂组成的机制。的 所涉及的因素将包括转录或翻译控制， 确定酶的水平和活性，竞争之间的作用， 不同糖基转移酶对共同底物的影响 糖基化酶和酶的亚细胞组织。最终 该信息可以形成改变抗原性的基础 肿瘤细胞的行为。

Gangliosides (sialylated glycosphingolipids) are ubiquitous components of mammalian cells. Ganglioside expression is altered in many tumor types, particularly in malignant melanoma, neuroblastoma, and astrocytoma. Gangliosides also serve as important antigens in these tumor types. While the pattern of expression of gangliosides in tumors is now well understood, very little is known about what determines the characteristic patterns in different cell types and the altered expression following transformation. The main goal of this project is to understand the ways that ganglioside expression in tumor cells is determined and regulated. The emphasis will be on an analysis of the various glycosyltransferases that are responsible for the biosynthesis of gangliosides in malignant melanoma and neuroblastoma cells. We have previously studied glycosyltransferase levels and activities in a panel of cells lines and identified key steps in the biosynthesis of gangliosides in these cells. GD3 synthase (SAT-2) and GM2\GD2 synthase (GalNAc-T) were particularly important enzymes in this process. We have recently isolated a cDNA clone coding for GalNAc-T and studied some of its properties. These studies will be extended by (i) continuing to characterize;, the GalNAc-T gene and its product, (ii) producing antibodies to this enzyme, (iii) using these antibodies to isolate the enzyme and to examine its glycosylation, (iv) attempt to clone a cDNA for SAT-2, (v) purify this enzyme and study its properties, and (vi) produce antibodies to SAT-2. Using these reagents we will be in a position to begin a more detailed analysis of the control mechanisms that determine the ganglioside makeup of these tumors. The factors involved will include transcriptional or translational control in determining enzyme levels and activity, the role of competition among the different glycosyltransferase for common substrates, the influence of glycosidases, and the sub-cellular organization of the enzymes. Ultimately this information could form the basis for altering the antigenicity or behavior of tumor cells.