Liang Yangui, Hua Qiang, Pan Pengwei, Yang Jie, Zhang Qi
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin, 300071, China.
Institute of Information On Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Biol Res. 2015 Sep 12;48(1):52. doi: 10.1186/s40659-015-0041-8.
Glycoproteins play a critical role in the cellular activities of eukaryotes. Sialic acid is typically the outermost monosaccharide of glycolipids and glycoproteins, and is necessary for normal development.
A strategy based on avidin-biotin affinity was established to enrich sialylated glycoproteins from HeLa cervical carcinoma, SW1990 pancreatic adenocarcinoma, and A549 lung adenocarcinoma cells. Using HPLC-MS/MS, western blot, real-time PCR, and enzyme-linked immunosorbent assay, gp96 was identified in all three cell lines. No significant difference in the protein expression of gp96 was detected at the whole cell level, but the amount of biotinylated gp96 in SW1990 cells was 30-40% lower than that in A549 and HeLa cells, and the amount of sialylated gp96 in SW1990 cells was 30% lower than that in A549 and HeLa cells. Immunoblotting results showed that the expression of sialyltransferase proteins in the total cell lysates from HeLa and A549 cells were higher than that in SW1990 cells.
We established a new method for investigating the expression and sialylation of glycoproteins using metabolic labeling, click chemistry, and avidin-biotin affinity. We successfully used this method to purify sialylated glycoproteins from cancer cell lines. Our results showed that the levels of gp96 sialylation varied across different cancer cell lines, and this may be because of differences in sialyltransferase expression.
糖蛋白在真核生物的细胞活动中起关键作用。唾液酸通常是糖脂和糖蛋白最外层的单糖，是正常发育所必需的。
建立了一种基于抗生物素蛋白-生物素亲和力的策略，用于从人宫颈癌HeLa细胞、胰腺腺癌SW1990细胞和肺腺癌A549细胞中富集唾液酸化糖蛋白。使用高效液相色谱-串联质谱、蛋白质免疫印迹、实时定量聚合酶链反应和酶联免疫吸附测定法，在所有这三种细胞系中均鉴定出gp96。在全细胞水平上未检测到gp96蛋白表达的显著差异，但SW1990细胞中生物素化gp96的量比A549和HeLa细胞低30%-40%，SW1990细胞中唾液酸化gp96的量比A549和HeLa细胞低30%。免疫印迹结果显示，HeLa和A549细胞总细胞裂解物中唾液酸转移酶蛋白的表达高于SW1990细胞。
我们建立了一种利用代谢标记、点击化学和抗生物素蛋白-生物素亲和力来研究糖蛋白表达和唾液酸化的新方法。我们成功地使用该方法从癌细胞系中纯化出唾液酸化糖蛋白。我们的结果表明，不同癌细胞系中gp96的唾液酸化水平不同，这可能是由于唾液酸转移酶表达的差异所致。
