Lizzi Anna Rita, D'Alessandro Anna Maria, Bozzi Argante, Cinque Benedetta, Oratore Arduino, D'Andrea Gabriele
Department of Biomedical Sciences and Technologies, University of L'Aquila, 67100 L'Aquila, Italy.
Mol Cell Biochem. 2007 Jun;300(1-2):29-37. doi: 10.1007/s11010-006-9343-z. Epub 2007 Apr 12.
The present paper shows that human chronic myeloid (K562) cells exposed 3 h to 20 microM 3'-azido-3'-deoxythymidine (AZT) exhibit marked variations of the oligosaccharide moiety of glycoconjugates. These changes were analyzed by confocal fluorescence microscopy, upon incubation of control and AZT-treated cells with biotin-lectin conjugates to visualize cell surface glycans or total glycans after cells permeabilization. In addition, cell fluorescence distribution of the biotinylated lectins, localized with streptavidin conjugates labeled with Alexa Fluor 488, was analyzed by flow cytometry. The results obtained show significant variations on the expression/distribution of membrane surface glycans as detected by both WGA and SNA, two lectins that recognize primarily cellular internal membrane glycolipids. A further interesting result was the significant increase of N-acetylglucosamine linked glycans localized either at the cell surface or intracellularly but only in K562 cells exposed to AZT. On the whole, our data demonstrate that AZT alters both lipid and N-linked glycosylations thus confirming previous observations, from our laboratory and from other Authors, that the drug impair the nucleotide-sugar import in the Golgi's lumen. AZT does also alter the O-linked glycosylations that occur in the Golgi complex since these reactions require the incorporation of sialic acid, GlcNAc and GalNAc all of which are sensitive to the drug.
本文表明，人慢性髓性（K562）细胞暴露于20微摩尔3'-叠氮基-3'-脱氧胸苷（AZT）3小时后，糖缀合物的寡糖部分表现出明显变化。在用生物素-凝集素缀合物孵育对照细胞和经AZT处理的细胞后，通过共聚焦荧光显微镜分析这些变化，以在细胞通透后可视化细胞表面聚糖或总聚糖。此外，通过流式细胞术分析用Alexa Fluor 488标记的链霉亲和素缀合物定位的生物素化凝集素的细胞荧光分布。所获得的结果表明，通过WGA和SNA这两种主要识别细胞内膜糖脂的凝集素检测到，膜表面聚糖的表达/分布存在显著变化。另一个有趣的结果是，仅在暴露于AZT的K562细胞中，位于细胞表面或细胞内的N-乙酰葡糖胺连接聚糖显著增加。总体而言，我们的数据表明AZT改变了脂质和N-连接糖基化，从而证实了我们实验室和其他作者先前的观察结果，即该药物损害了高尔基体腔中核苷酸糖的导入。AZT还会改变在高尔基体复合物中发生的O-连接糖基化，因为这些反应需要掺入唾液酸、GlcNAc和GalNAc，而所有这些都对该药物敏感。
