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Fluorescein-5-thiosemicarbazide as a probe for directly imaging of mucin-type O-linked glycoprotein within living cells

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Abstract

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 Ac4GalNAc 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.

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Abbreviations

FTSC:

Fluorescein-5-thiosemicarbazide

DAPI:

4′,6-diamidino-2-phenylindole

DMSO:

Dimethyl sulfoxide

keto sugar:

2-keto Ac4GalNAc analogue

FITC:

Fluorescein isothiocyanate

ESI-MS:

Electrospray ionization mass spectrometry

LC-MS:

Liquid chromatography mass spectrometry

HPLC:

High performance liquid chromatography

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Acknowledgments

This work was supported by National Natural Science Foundation of China (NSFC) (30870548, 31071506, 31170773), Foundation of Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education in Northwest University (KH09019) and Program for New Century Excellent Talents in Universities (NCET-08-0893).

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Authors and Affiliations

  1. Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, Xi’an, 710069, People’s Republic of China

    Ying Zhang, Yujiao Sun, Zhongfu Wang & Linjuan Huang

  2. College of Life Sciences, Northwest University, 229 Northern Taibai Road, Xi’an, 710069, People’s Republic of China

    Zhongfu Wang & Linjuan Huang

Authors
  1. Ying Zhang
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  2. Yujiao Sun
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  3. Zhongfu Wang
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  4. Linjuan Huang
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Correspondence to Zhongfu Wang or Linjuan Huang.

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Zhang, Y., Sun, Y., Wang, Z. et al. Fluorescein-5-thiosemicarbazide as a probe for directly imaging of mucin-type O-linked glycoprotein within living cells. Glycoconj J 29, 445–452 (2012). https://doi.org/10.1007/s10719-012-9425-y

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  • DOI: https://doi.org/10.1007/s10719-012-9425-y

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