A novel strategy for mammalian cell surface glycome profiling using lectin microarray
A novel strategy for mammalian cell surface glycome profiling using lectin microarray
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DOI：
10.1093/glycob/cwm084
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发表时间：
2007-10-01
期刊：
影响因子：
4.3
通讯作者：
Hirabayashi, Jun
中科院分区：
文献类型：
--
作者：
Tateno, Hiroaki;Uchiyama, Noboru;Hirabayashi, Jun
多糖代表细胞中表达的多糖的总集合。糖被认为在细胞类型、发育和分化阶段以及恶性转化过程中变化。对糖聚糖的分析为了解糖聚糖在这些细胞过程中的功能提供了基础。最近，一种被称为凝集素微阵列的技术被开发用于糖基化的快速分析，尽管它的使用主要局限于细胞裂解物的糖蛋白，因此无法分析完整的细胞表面聚糖。在这里，我们报告了一种基于该技术的简单而敏感的程序，用于直接分析活的哺乳动物细胞表面糖。将荧光标记的活细胞原位应用于建立的由43种具有不同结合特异性的固定凝集素组成的凝集素微阵列。洗涤后，结合的细胞在不固定和不渗透的情况下，在液相中直接用倏逝场荧光扫描仪检测。通过对CHO及其糖基化缺陷突变细胞、野生型和β 1-3- n-乙酰氨基葡萄糖转移酶II敲除小鼠的脾细胞进行差异谱分析获得的模型实验结果与它们的糖基化表型非常吻合。我们还比较了K562细胞分化前后的细胞表面聚糖，发现分化细胞上o -聚糖的表达明显增加。这些结果表明，该技术为分析哺乳动物细胞表面糖的全局变化提供了一种新的策略。
The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wildtype and beta 1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.