Fluorescein-5-thiosemicarbazide as a probe for directly imaging of mucin-type O-linked glycoprotein within living cells.
Mucin-type O-linked glycoproteins are known for regulating many aspects of cell activity but remains a challenge to detect under physiological conditions which is due to the diversity of O-glycosylation and the lack of universal method. Here a direct labeling strategy for in situ visualizing of mucin-type O-linked glycoproteins on living cells has been developed. The strategy utilizes the combination of metabolic engineering and chemical probing technologies. Treating cells with an unnatural sugar, 2-keto Ac(4)GalNAc analogue (2-keto isostere of GalNAc) to generate keto groups upon cells, followed by chemoselective ligation of keto groups on cells with a fluorescent tag, fluorescein-5-thiosemicarbazide (FTSC), provides a promising platform to probing mucin-type O-glycosylation on living cells. The FTSC conjugates illustrated very similar fluorescent spectra as FITC, a fluorescent tag widely used in proteomics, indicating good compatibility with commonly used fluorescent equipments. The established method eliminated the need of an additional fluorescent amplification step. Cells after being treated with the method maintained a rather high level of viability of 84.3%. Finally, the assay has been successfully applied to image the expression of mucin-type O-linked glycoproteins within CHO and HeLa cells.
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, Xi'an, People's Republic of China.
Ying
Y
Zhang
Yujiao
Y
Sun
Zhongfu
Z
Wang
Linjuan
L
Huang
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D009077:Mucins / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling / Q000379:methods
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009077:Mucins; C027912:fluorescein thiosemicarbazide; D000116:Acetylgalactosamine