Glycoproteomics and glycomics investigation of membrane N‐glycosylproteins from human colon carcinoma cells
Glycoproteomics and glycomics investigation of membrane N‐glycosylproteins from human colon carcinoma cells
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DOI：
10.1002/pmic.200800151
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发表时间：
2008-08
期刊：
影响因子：
3.4
通讯作者：
A. Vercoutter‐Edouart;M. Slomianny;Olivia Dekeyzer-Beseme;J. Haeuw;J. Michalski
中科院分区：
文献类型：
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作者：
A. Vercoutter‐Edouart;M. Slomianny;Olivia Dekeyzer-Beseme;J. Haeuw;J. Michalski
已知蛋白质的异常糖基化深刻地影响肿瘤细胞的细胞粘附或运动性。在这项研究中，我们使用HT-29人结肠上皮癌细胞作为癌症进展的细胞模型，因为它们可以增殖或分化成肠上皮细胞表型。基于Con A凝集素亲和层析、SDS-PAGE和MS分析的糖蛋白组学方法，可以从膜制备的Triton X-100增溶蛋白中鉴定膜N-糖蛋白。其中，65%是膜蛋白，45%已知是N-糖基化的，如α链整合素和二肽基异构酶IV。通过凝集素印迹分析，在增殖和分化的HT-29细胞之间观察到膜糖蛋白的α-2，3-和α-2，6-唾液酸化的显著变化。根据这些结果，对相应条带的胰蛋白酶进行了nano-LC-MS/MS分析，并鉴定了几种跨膜糖蛋白，如溶质载体家族成员和粘附蛋白。最后，我们使用全甲基化衍生物的MALDI-MS和GC-MS分析比较了增殖和肠上皮细胞样HT-29细胞的N-聚糖谱和单糖组成。这种糖组学方法可以强调N-聚糖结构的显著变化，特别是肠上皮细胞样HT-29细胞中非典型N-乙酰葡糖胺（GlcNAc）末端N-聚糖的表达。
Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT‐29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin‐affinity chromatography, SDS‐PAGE and MS analysis, allowed the identification of membrane N‐glycoproteins from Triton X‐100‐solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N‐glycosylated, such as α chains integrin and dipeptidyl isomerase IV. By lectin‐blot analysis, significant changes of α‐2,3‐ and α‐2,6‐sialylation of membrane glycoproteins were observed between proliferating and differentiated HT‐29 cells. From these results, nano‐LC‐MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N‐glycans profiles and monosaccharide composition of proliferating and enterocyte‐like HT‐29 cells using MALDI‐MS and GC‐MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N‐glycans structure, in particular the expression of atypical N‐acetylglucosamine (GlcNAc)‐ended N‐glycans in enterocyte‐like HT‐29 cells.