Chemoenzymatic Labeling, Detection and Profiling of Core Fucosylation in Live Cells

Creators: Zhu, Qiang¹; Chaubard, Jean-Luc; Geng, Didi¹; Shen, Jiechen²; Ban, Lan; Cheung, Sheldon T.; Wei, Fangyu³; Liu, Yating³; Sun, Haofan⁴; Calderon, Angie⁵; Dong, Wenbo²; Qin, Weijie⁴; Li, Tiehai³; Wen, Liuqing³; Wang, Peng George⁵; Sun, Shisheng²; Yi, Wen (Corresponding)¹; Hsieh-Wilson, Linda C. (Corresponding)⁶

1. Zhejiang University
2. Northwest University
3. Shanghai Institute of Materia Medica
4. Beijing Proteome Research Center
5. Southern University of Science and Technology
6. California Institute of Technology

Abstract

Core fucosylation, the attachment of an α-1,6-linked-fucose to the N-glycan core pentasaccharide, is an abundant protein modification that plays critical roles in various biological processes such as cell signaling, B cell development, antibody-dependent cellular cytotoxicity, and oncogenesis. However, the tools currently used to detect core fucosylation suffer from poor specificity, exhibiting cross-reactivity against all types of fucosylation. Herein we report the development of a new chemoenzymatic strategy for the rapid and selective detection of core fucosylated glycans. This approach employs a galactosyltransferase enzyme identified fromCaenorhabditis elegansthat specifically transfers an azido-appended galactose residue onto core fucose via a β-1,4 glycosidic linkage. We demonstrate that the approach exhibits superior specificity toward core fucose on a variety of complex N-glycans. The method enables detection of core fucosylated glycoproteins from complex cell lysates, as well as on live cell surfaces, and it can be integrated into a diagnostic platform to profile protein-specific core fucosylation levels. This chemoenzymatic labeling approach offers a new strategy for the identification of disease biomarkers and will allow researchers to further characterize the fundamental role of this important glycan in normal and disease physiology.

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Contributions

Q.Z. and J.-L.C. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgement

We are grateful to Prof. Markus Aebi (ETH Zurich, Switzerland) for providing the GALT-1 plasmid, Prof. Iain Wilson (BOKU University, Vienna, Austria) for the core fucosylated glycopeptide and Prof. Jianguo Gu (Tohoku Pharmaceutical University, Sendai, Japan) for the Fut8 knockout samples. This work was supported by grants from the National Institutes of Health (R01 AG060540 and AG062324 to L.C.H.-W.), the National Natural Science Foundation of China (NSFC, grant nos. 22325704, 32271331, 92353303 to W.Y., 32201045, 32471327 to Q.Z., and 22007092 to L.W.), the Lingang Laboratory (LG-QS-202206-08 to L.W.), the Fundamental Research Funds for Central Universities (K20220228 to W.Y.).

Additional Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c09303.

Experimental procedures, supplementary schemes and figures, and NMR spectra (PDF)
Intact glycopeptides identified from control raw file and GalT-catalyzed raw file using StrucGP (XLSX)
Core fucosylated proteins based on GalT labeling method (XLSX)

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