Development of a novel method to evaluate sialylation of glycoproteins and analysis of gp96 sialylation in Hela, SW1990 and A549 cell lines

Biological research: BR

Qiang Hua¹  Jie Yang²  Pengwei Pan²  Yangui Liang²  Qi Zhang² 

[1] Institute of Information On Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China;State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin, China

关键词: Click chemistry;    Sialic acid;    gp96;    Sialyltransferase;   

DOI  :  10.1186/s40659-015-0041-8

学科分类：生物科学（综合）

来源: BioMed Central

【 摘 要 】

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.

【 授权许可】

CC BY