Spielman J, Hull S R, Sheng Z Q, Kanterman R, Bright A, Carraway K L
Department of Anatomy and Cell Biology, University of Miami School of Medicine, Florida 33101.
J Biol Chem. 1988 Jul 15;263(20):9621-9.
The major cell surface glycoprotein (ascites sialoglycoprotein-1 (ASGP-1] of ascites 13762 rat mammary tumor cells is a large (Mr greater than 500,000), highly glycosylated sialomucin which is present in great abundance (greater than 0.5% of total cell protein). Thus, these tumors provide a useful system for investigating the biosynthesis of O-glycosylated glycoproteins. Previous studies in this system have demonstrated that initiation of O-linked oligosaccharides occurs throughout most of the transit period of ASGP-1 from the endoplasmic reticulum to the cell surface. By pulse-chase threonine labeling and precipitation with peanut agglutinin, ASGP-1 is first observed as an immature lightly glycosylated form (Mr approximately 200,000) which is converted to a more mature, more heavily glycosylated form (designated the premature or P form) with a half-time of about 30 min. The P form is then more gradually converted into the mature ASGP-1. Analysis of glucosamine-labeled oligosaccharitols obtained from the immature form showed primarily unsialylated derivatives consisting of the structures of the size of the tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc and smaller, whereas the mature form showed a mixture of sialylated and unsialylated structures. Desialylation of glucosamine-labeled mature form resulted in a glycoprotein intermediate in size between the immature and mature forms, indicating that the size change with maturation is not solely due to sialylation. Treatment of the cells with 10(-6) M monensin significantly reduced the conversion of immature to mature form without inhibiting initiation of O-linked oligosaccharides and without preventing sialylation. Analysis of oligosaccharitols obtained from ASGP-1 of monensin-treated cells showed that the major oligosaccharides are trisaccharide GlcNAc beta 1,6(Gal beta 1,3)GalNAc and sialylated trisaccharide GlcNAc beta 1,6(NeuAc alpha 2,3-Gal-beta 1,3) GalNAc. These results suggest that monensin specifically disrupts the compartment of the biosynthetic pathway which adds most of the beta 1,4-Gal to the oligosaccharides of ASGP-1 and that this compartment is separate from the primary site of sialylation.
腹水13762大鼠乳腺肿瘤细胞的主要细胞表面糖蛋白（腹水唾液酸糖蛋白-1，ASGP-1）是一种大分子（分子量大于500,000）、高度糖基化的唾液粘蛋白，其含量丰富（大于细胞总蛋白的0.5%）。因此，这些肿瘤为研究O-糖基化糖蛋白的生物合成提供了一个有用的系统。此前在该系统中的研究表明，O-连接寡糖的起始发生在ASGP-1从内质网到细胞表面的大部分转运过程中。通过脉冲追踪苏氨酸标记并用花生凝集素沉淀，ASGP-1首先以未成熟的轻度糖基化形式（分子量约200,000）被观察到，该形式会在约30分钟的半衰期内转化为更成熟、糖基化程度更高的形式（称为早熟或P形式）。然后P形式会更逐渐地转化为成熟的ASGP-1。对从未成熟形式获得的氨基葡萄糖标记的低聚糖醇的分析表明，主要是由四糖Galβ1,4GlcNAcβ1,6（Galβ1,3）GalNAc及更小尺寸结构组成的非唾液酸化衍生物，而成熟形式则显示出唾液酸化和非唾液酸化结构的混合物。对氨基葡萄糖标记的成熟形式进行脱唾液酸处理会产生一种分子量介于未成熟和成熟形式之间的糖蛋白中间体，这表明随着成熟发生的尺寸变化并非仅仅是由于唾液酸化。用10^(-6) M莫能菌素处理细胞会显著降低未成熟形式向成熟形式的转化，而不抑制O-连接寡糖的起始，也不阻止唾液酸化。对从经莫能菌素处理的细胞的ASGP-1中获得的低聚糖醇的分析表明，主要的低聚糖是三糖GlcNAcβ1,6（Galβ1,3）GalNAc和唾液酸化三糖GlcNAcβ1,6（NeuAcα2,3-Gal-β1,3）GalNAc。这些结果表明，莫能菌素特异性地破坏了生物合成途径中向ASGP-1的寡糖添加大部分β1,4-半乳糖的区室，并且该区室与唾液酸化的主要位点是分开的。
