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Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide

Abstract

Introduction

The promise of the natural immunoregulator, Galectin-1 (Gal1), as an immunomodulatory therapeutic is challenged by its unstable homodimeric conformation. Previously, a Gal1 homodimer stabilized via covalent poly(ethylene glycol) diacrylate (PEGDA) cross-linking demonstrated higher activity relative to the non-covalent homodimer.

Methods

Here, we report Gal1 homodimers formed using an alternative thiol-Michael addition linker chemistry.

Results

Poly(ethylene glycol) bismaleimide (PEGbisMal) reacted with Gal1 at multiple sites with greater efficiency than PEGDA. However, multiple PEGbisMal molecules were conjugated to Gal1 C130, a Gal1 mutant with one surface cysteine (cys-130) and two cysteines thought to be buried in the solvent-inaccessible protein core (cys-42 and cys-60). Site-directed mutagenesis demonstrated that cys-60 was the site at which additional PEGbisMal molecules were conjugated onto Gal1 C130. Compared to WT-Gal1, Gal1 C130 had low activity for inducing Jurkat T cell death, characterized by phosphatidylserine exposure and membrane permeability. PEG cross-linking could restore the function of Gal1 C130, such that at high concentrations Gal1 C130 cross-linked by PEGbisMal had higher activity than both WT-Gal1 and Gal1 C130 cross-linked by PEGDA. Mutating cys-42 and cys-60 to serines in Gal1 C130 did not affect the cell death signaling activity of the Gal1 C130 homodimer cross-linked by PEGbisMal. PEGylated Gal1 C130 variants also eliminated the need for a reducing agent, such as dithiothreitol, which is required to maintain WT-Gal1 signaling activity.

Conclusion

Collectively, these data demonstrate that thiol-Michael addition bioconjugation leads to a PEG-cross-linked Gal1 homodimer with improved extracellular signaling activity that does not require a reducing environment to be functional.

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Acknowledgments

This research was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under University of Florida Clinical and Translational Science Award TL1TR001428, the National Science Foundation grant DMR-1455201, and start-up funds from the University of Florida. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, nor the National Science Foundation.

Conflict of interest

Bryant J. Kane, Margaret M. Fettis, Shaheen A. Farhadi, Renjie Liu declare that they have no conflicts of interest. Gregory A. Hudalla is a founder of Anchor Biologics, Inc.

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No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

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Correspondence to Gregory A. Hudalla.

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Kane, B.J., Fettis, M.M., Farhadi, S.A. et al. Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide. Cel. Mol. Bioeng. 14, 523–534 (2021). https://doi.org/10.1007/s12195-021-00681-0

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  • DOI: https://doi.org/10.1007/s12195-021-00681-0

Keywords

  • Protein engineering
  • Bioconjugation
  • Galectin
  • Protein-polymer conjugate
  • Protein dimerization