De Novo Glycan Display on Cell Surfaces Using HaloTag: Visualizing the Effect of the Galectin Lattice on the Lateral Diffusion and Extracellular Vesicle Loading of Glycosylated Membrane Proteins

De Novo Glycan Display on Cell Surfaces Using HaloTag: Visualizing the Effect of the Galectin Lattice on the Lateral Diffusion and Extracellular Vesicle Loading of Glycosylated Membrane Proteins

Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecu...

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Bibliographic Details
Published in:Journal of the American Chemical Society Vol. 146; no. 32; pp. 22193 - 22207
Main Authors: Miura, Ayane, Manabe, Yoshiyuki, Suzuki, Kenichi G. N., Shomura, Hiroki, Okamura, Soichiro, Shirakawa, Asuka, Yano, Kumpei, Miyake, Shuto, Mayusumi, Koki, Lin, Chun-Cheng, Morimoto, Kenta, Ishitobi, Jojiro, Nakase, Ikuhiko, Arai, Kenta, Kobayashi, Shouhei, Ishikawa, Ushio, Kanoh, Hirotaka, Miyoshi, Eiji, Yamaji, Toshiyuki, Kabayama, Kazuya, Fukase, Koichi
Format: Journal Article
Language:English
Published: United States American Chemical Society 14-08-2024
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Summary:Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecules. Glycocalyx-editing techniques offer potent tools to probe their functions. In this study, we devised a HaloTag-based technique for glycan manipulation, which enables the introduction of chemically synthesized glycans onto a specific protein (protein of interest, POI) and concurrently incorporates fluorescent units to attach homogeneous, well-defined glycans to the fluorescence-labeled POIs. Leveraging this HaloTag-based glycan-display system, we investigated the influence of the interactions between Gal-3 and various N-glycans on protein dynamics. Our analyses revealed that glycosylation modulates the lateral diffusion of the membrane proteins in a structure-dependent manner through interaction with Gal-3, particularly in the context of the Gal-3-induced formation of the glycan network (galectin lattice). Furthermore, N-glycan attachment was also revealed to have a significant impact on the extracellular vesicle-loading of membrane proteins. Notably, our POI-specific glycan introduction does not disrupt intact glycan structures, thereby enabling a functional analysis of glycans in the presence of native glycan networks. This approach complements conventional glycan-editing methods and provides a means for uncovering the molecular underpinnings of glycan functions on the cell surface.
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ISSN:0002-7863
1520-5126
1520-5126
DOI:10.1021/jacs.4c02040