Iozzo R V, Clark C C
J Biol Chem. 1987 Aug 15;262(23):11188-99.
We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.
我们研究了在无L-谷氨酰胺或有谷氨酰胺类似物6-重氮-5-氧代-L-正亮氨酸（DON）存在的情况下培养的人结肠癌细胞中硫酸乙酰肝素蛋白聚糖的生物合成。在用100微克/毫升DON（0.58毫摩尔）孵育24小时或无L-谷氨酰胺的情况下，[35S]硫酸盐的掺入被最大程度地抑制至约50%，而[3H]亮氨酸或[3H]丝氨酸的掺入及其比活性没有受到显著影响。几条证据表明，[35S]硫酸盐掺入的抑制是由UDP-N-乙酰己糖胺细胞内池的减少介导的：细胞内己糖胺水平降低了约50%；考虑到比活性的变化，[3H]葡萄糖胺的掺入也被显著抑制；外源性D-葡萄糖胺（2.3毫摩尔）能够基本上逆转DON的抑制作用。内源性糖供应的这种减少导致产生了一种糖基化不足、浮力密度较低的蛋白聚糖，其硫酸乙酰肝素链的数量明显减少，大小和硫酸化程度正常，O-连接寡糖的数量甚至更少。细胞表面出现较小的钌红染色蛋白聚糖，从超微结构上证实了这些生化变化。DON还显著抑制细胞增殖并引起深刻的形态学变化，在无DON的培养基中培养时，这两种变化都是可逆的。这些结果表明，谷氨酰胺代谢的紊乱对硫酸乙酰肝素蛋白聚糖的结构和人结肠癌细胞的表型有深远影响，并表明DON处理可用于研究各种细胞系统中蛋白聚糖的翻译后修饰。
