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Genetically encoded chemical crosslinking of carbohydrate

Nature Chemistry volume 15, pages 33–42 (2023)Cite this article

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Abstract

Protein–carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins. SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein–sugar interactions.

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Fig. 1: SF was identified as a suitable functional group for crosslinking carbohydrate through proximity-enabled reactivity using a strategy that involves plant-and-cast small-molecule crosslinkers.
Fig. 2: Crosslinking of Siglec-7v with azido-GD3 by NHSF was dependent on the concentration and the specific protein–carbohydrate binding.
Fig. 3: Crosslinking site on Siglec-7v and distance dependence of the crosslinker indicate that the SF of NHSF reacted with the carbohydrate via proximity-enabled reactivity.
Fig. 4: Genetic incorporation of SFY into proteins in E. coli.
Fig. 5: Siglec-7v(SFY) crosslinked with azido-GD3 in vitro and with sialoglycan on the cell surface.
Fig. 6: Siglec-7v(SFY)-enhanced NK cell killing of cancer cells.

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Data availability

All data supporting the results and conclusions are available within the article and its Supplementary Information. Synthetic experimental procedures, compound characterization, NMR and HPLC are available in the Supplementary Information. Requests for materials should be addressed to L.W. Source data are provided with this paper.

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Acknowledgements

L.W. acknowledges the support of the NIH (R01GM118384 and R01CA258300).

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  1. These authors contributed equally: Shanshan Li, Nanxi Wang, Bingchen Yu.

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA

    Shanshan Li, Nanxi Wang, Bingchen Yu, Wei Sun & Lei Wang

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Contributions

S.L. and L.W. conceived research; S.L., B.Y. and L.W. designed the experiments; S.L., N.W., B.Y. and W.S. performed the experiments; S.L. and L.W. analysed the data; S.L., B.Y. and L.W. wrote the manuscript; L.W. supervised the project; and all the authors read and approved the manuscript.

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Correspondence to Lei Wang.

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Competing interests

L.W., S.L. and N.W. are inventors on a patent application filed as International Application No. PCT/US2022/031925 by The Regents of the University of California. The patent application covers the unnatural amino acid SFY and its ability to crosslink carbohydrates, as described in the article. The other authors (B.Y. and W.S.) declare no competing interests.

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Nature Chemistry thanks Ryan Flynn, Stephen Fried and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Chemo-enzymatic synthesis of azido-GD3, chemical synthesis procedure, NMR spectra and Supplementary Figs. 1–9, Tables 1 and 2 and Data Files.

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Unprocessed western blots and statistical source data.

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Unprocessed western blots and statistical source data.

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Li, S., Wang, N., Yu, B. et al. Genetically encoded chemical crosslinking of carbohydrate. Nat. Chem. 15, 33–42 (2023). https://doi.org/10.1038/s41557-022-01059-z

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