Hull S R, Carraway K L
Department of Anatomy and Cell Biology, University of Miami School of Medicine, Florida 33101.
J Cell Biochem. 1989 May;40(1):67-81. doi: 10.1002/jcb.240400108.
ASGP-1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O-linked oligosaccharide chains. We have used this system to investigate the O-glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S-1 and S-2) were isolated as their oligosaccharitols by alkaline borohydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide -O4S-GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and S-2 its sialylated derivative -O4S-GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP-1 intermediate in size between immature and mature sialomucin. Pulse-chase analyses showed that the intermediate could be chased into mature ASGP-1. The concomitant conversion of S-1 into S-2 had a half-time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and sialylated derivative GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNAc. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with beta-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as beta-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.
ASGP-1是13762腹水大鼠乳腺腺癌的主要细胞表面涎酸粘蛋白，至少占腹水细胞总蛋白的0.5%，其20%的O-连接寡糖链上带有硫酸根。我们利用该系统研究了这些细胞中的O-糖基化途径，并确定了硫酸化和唾液酸化之间的时间关系。通过碱性硼氢化物消除、阴离子交换高效液相色谱和离子抑制高效液相色谱，将两种主要的硫酸化寡糖（S-1和S-2）分离为它们的寡糖醇。结构分析表明，S-1是一种分支的硫酸化三糖-O4S-GlcNAcβ1,6-(Galβ1,3)-GalNAc，S-2是其唾液酸化衍生物-O4S-GlcNAcβ1,6-(NeuAcα2,3-Galβ1,3)-GalNac。用硫酸盐进行脉冲标记表明，硫酸化主要发生在大小介于未成熟和成熟涎酸粘蛋白之间的一种ASGP-1中间体上。脉冲追踪分析表明，该中间体可以追踪到成熟的ASGP-1中。S-1同时转化为S-2的半衰期不到5分钟。用莫能菌素处理肿瘤细胞导致硫酸化受到95%的抑制，未硫酸化的三糖GlcNAcβ1,6-(Galβ1,3)-GalNAc和唾液酸化衍生物GlcNAcβ1,6-(NeuAcα2,3-Galβ1,3)-GalNAc积累。这些数据表明，ASGP-1的硫酸化是一个中间合成步骤，它与β-1,4-半乳糖基化竞争三糖中间体，因此与β-1,4-半乳糖基化发生在同一区室。此外，硫酸化先于唾液酸化，但在ASGP-1的寡糖组装中，两者是快速连续的动力学事件。
