Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Baldwin, A. D., and K. L. Kiick. Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjug. Chem. 22:1946–1953, 2011.

    Article  Google Scholar 

  2. Barrientos, G., N. Freitag, I. Tirado-González, L. Unverdorben, U. Jeschke, V. L. J. L. Thijssen, and S. M. Blois. Involvement of galectin-1 in reproduction: past, present and future. Hum. Reprod. Update 20:175–193, 2014.

    Article  Google Scholar 

  3. Camby, I., M. Le Mercier, F. Lefranc, and R. Kiss. Galectin-1: a small protein with major functions. Glycobiology 16:137R–157R, 2006.

    Article  Google Scholar 

  4. Cedeno-Laurent, F., S. R. Barthel, M. J. Opperman, D. M. Lee, R. A. Clark, and C. J. Dimitroff. Development of a nascent galectin-1 chimeric molecule for studying the role of leukocyte galectin-1 ligands and immune disease modulation. J. Immunol. 185:4659–4672, 2010.

    Article  Google Scholar 

  5. Chien, C.-T. H., M.-R. Ho, C.-H. Lin, and S.-T. D. Hsu. Lactose binding induces opposing dynamics changes in human galectins revealed by NMR-based hydrogen–deuterium exchange. Molecules 22(8):1357, 2017.

    Article  Google Scholar 

  6. Earl, L. A., S. Bi, and L. G. Baum. Galectin multimerization and lattice formation are regulated by linker region structure. Glycobiology 21:6–12, 2011.

    Article  Google Scholar 

  7. Elbert, D. L., and J. A. Hubbell. Conjugate addition reactions combined with free-radical cross-linking for the design of materials for tissue engineering. Biomacromolecules 2:430–441, 2001.

    Article  Google Scholar 

  8. Farhadi, S. A., M. M. Fettis, R. Liu, and G. A. Hudalla. A synthetic tetramer of galectin-1 and galectin-3 amplifies pro-apoptotic signaling by integrating the activity of both galectins. Front. Chem. 7:898, 2020.

    Article  Google Scholar 

  9. Farhadi, S. A., and G. A. Hudalla. Engineering galectin–glycan interactions for immunotherapy and immunomodulation. Exp. Biol. Med. 241:1074–1083, 2016.

    Article  Google Scholar 

  10. Fettis, M. M., and G. A. Hudalla. Engineering reactive oxygen species-resistant galectin-1 dimers with enhanced lectin activity. Bioconjug. Chem. 29:2489–2496, 2018.

    Article  Google Scholar 

  11. Fontaine, S. D., R. Reid, L. Robinson, G. W. Ashley, and D. V. Santi. Long-term stabilization of maleimide–thiol conjugates. Bioconjug. Chem. 26:145–152, 2015.

    Article  Google Scholar 

  12. Guardia, C. M., J. J. Caramelo, M. Trujillo, S. P. Méndez-Huergo, R. Radi, D. A. Estrin, and G. A. Rabinovich. Structural basis of redox-dependent modulation of galectin-1 dynamics and function. Glycobiology 24:428–441, 2014.

    Article  Google Scholar 

  13. Hong, L., Z. Wang, X. Wei, J. Shi, and C. Li. Antibodies against polyethylene glycol in human blood: a literature review. J. Pharmacol. Toxicol. Methods 102:2020.

    Article  Google Scholar 

  14. Huang, W., X. Wu, X. Gao, Y. Yu, H. Lei, Z. Zhu, Y. Shi, Y. Chen, M. Qin, W. Wang, and Y. Cao. Maleimide–thiol adducts stabilized through stretching. Nat. Chem. 11:310–319, 2019.

    Article  Google Scholar 

  15. Hudalla, G. A., T. S. Eng, and W. L. Murphy. An approach to modulate degradation and mesenchymal stem cell behavior in poly(ethylene glycol) networks. Biomacromolecules 9:842–849, 2008.

    Article  Google Scholar 

  16. Ito, K., K. Stannard, E. Gabutero, A. M. Clark, S.-Y. Neo, S. Onturk, H. Blanchard, and S. J. Ralph. Galectin-1 as a potent target for cancer therapy: role in the tumor microenvironment. Cancer Metastasis Rev. 31:763–778, 2012.

    Article  Google Scholar 

  17. Ko, J. H., and H. D. Maynard. A guide to maximizing the therapeutic potential of protein–polymer conjugates by rational design. Chem. Soc. Rev. 47:8998–9014, 2018.

    Article  Google Scholar 

  18. Koniev, O., and A. Wagner. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44:5495–5551, 2015.

    Article  Google Scholar 

  19. Lorenzo, M. M., C. G. Decker, M. U. Kahveci, S. J. Paluck, and H. D. Maynard. Homodimeric protein-polymer conjugates via the tetrazine-trans-cyclooctene ligation. Macromolecules 49:30–37, 2016.

    Article  Google Scholar 

  20. Miura, T., M. Takahashi, H. Horie, H. Kurushima, D. Tsuchimoto, K. Sakumi, and Y. Nakabeppu. Galectin-1β, a natural monomeric form of galectin-1 lacking its six amino-terminal residues promotes axonal regeneration but not cell death. Cell Death Differ. 11:1076–1083, 2004.

    Article  Google Scholar 

  21. Nair, D. P., M. Podgórski, S. Chatani, T. Gong, W. Xi, C. R. Fenoli, and C. N. Bowman. The thiol-michael addition click reaction: a powerful and widely used tool in materials chemistry. Chem. Mater. 26:724–744, 2014.

    Article  Google Scholar 

  22. Nesmelova, I. V., E. Ermakova, V. A. Daragan, M. Pang, M. Menéndez, L. Lagartera, D. Solís, L. G. Baum, and K. H. Mayo. Lactose binding to galectin-1 modulates structural dynamics, increases conformational entropy, and occurs with apparent negative cooperativity. J Mol Biol 397:1209–1230, 2010.

    Article  Google Scholar 

  23. Nishi, N., A. Abe, J. Iwaki, H. Yoshida, A. Itoh, H. Shoji, S. Kamitori, J. Hirabayashi, and T. Nakamura. Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1. Glycobiology 18:1065–1073, 2008.

    Article  Google Scholar 

  24. Nishi, N., A. Itoh, A. Fujiyama, N. Yoshida, S. Araya, M. Hirashima, H. Shoji, and T. Nakamura. Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins. FEBS Lett. 579:2058–2064, 2005.

    Article  Google Scholar 

  25. Odom, O. W., W. Kudlicki, G. Kramer, and B. Hardesty. An effect of polyethylene glycol 8000 on protein mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and a method for eliminating this effect. Anal. Biochem. 245:249–252, 1997.

    Article  Google Scholar 

  26. Pace, K. E., H. P. Hahn, and L. G. Baum. Preparation of recombinant human galectin-1 and use in T cell death assays. Methods Enzymol. 363:499–518, 2003.

    Article  Google Scholar 

  27. Pelegri-O’Day, E. M., E.-W. Lin, and H. D. Maynard. Therapeutic protein–polymer conjugates: advancing beyond PEGylation. J. Am. Chem. Soc. 136:14323–14332, 2014.

    Article  Google Scholar 

  28. Ravasco, J. M. J. M., H. Faustino, A. Trindade, and P. M. P. Gois. Bioconjugation with maleimides: a useful tool for chemical biology. Chem. Eur. J. 25:43–59, 2019.

    Article  Google Scholar 

  29. Schellekens, H., W. E. Hennink, and V. Brinks. The immunogenicity of polyethylene glycol: facts and fiction. Pharm. Res. 30:1729–1734, 2013.

    Article  Google Scholar 

  30. Shen, B.-Q., K. Xu, L. Liu, H. Raab, S. Bhakta, M. Kenrick, K. L. Parsons-Reponte, J. Tien, S.-F. Yu, E. Mai, D. Li, J. Tibbitts, J. Baudys, O. M. Saad, S. J. Scales, P. J. McDonald, P. E. Hass, C. Eigenbrot, T. Nguyen, W. A. Solis, R. N. Fuji, K. M. Flagella, D. Patel, S. D. Spencer, L. A. Khawli, A. Ebens, W. L. Wong, R. Vandlen, S. Kaur, M. X. Sliwkowski, R. H. Scheller, P. Polakis, and J. R. Junutula. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30:184–189, 2012.

    Article  Google Scholar 

  31. Sundblad, V., L. G. Morosi, J. R. Geffner, and G. A. Rabinovich. Galectin-1: a jack-of-all-trades in the resolution of acute and chronic inflammation. J. Immunol. 199:3721–3730, 2017.

    Article  Google Scholar 

  32. Tao, L., C. S. Kaddis, R. R. O. Loo, G. N. Grover, J. A. Loo, and H. D. Maynard. Synthetic approach to homodimeric protein-polymer conjugates. Chem. Commun. 16:2148–2150, 2009.

    Article  Google Scholar 

  33. Thijssen, V. L., and A. W. Griffioen. Galectin-1 and -9 in angiogenesis: a sweet couple. Glycobiology 24:915–920, 2014.

    Article  Google Scholar 

  34. van der Leij, J., A. van den Berg, G. Harms, H. Eschbach, H. Vos, P. Zwiers, R. van Weeghel, H. Groen, S. Poppema, and L. Visser. Strongly enhanced IL-10 production using stable galectin-1 homodimers. Mol. Immunol. 44:506–513, 2007.

    Article  Google Scholar 

  35. White, C. J., and J. W. Bode. PEGylation and dimerization of expressed proteins under near equimolar conditions with potassium 2-pyridyl acyltrifluoroborates. ACS Cent. Sci. 4:197–206, 2018.

    Article  Google Scholar 

  36. Zhang, B., P. Chakma, M. P. Shulman, J. Ke, Z. A. Digby, and D. Konkolewicz. Probing the mechanism of thermally driven thiol-michael dynamic covalent chemistry. Org. Biomol. Chem. 16:2725–2734, 2001.

    Article  Google Scholar 

  37. Zheng, C. Y., G. Ma, and Z. Su. Native PAGE eliminates the problem of PEG–SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation. Electrophoresis 28:2801–2807, 2007.

    Article  Google Scholar 

Download references

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.

Ethical standards

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

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA

    Bryant J. Kane

  2. J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, 32611, USA

    Margaret M. Fettis, Shaheen A. Farhadi, Renjie Liu & Gregory A. Hudalla

  3. Biomedical Sciences J293, PO BOX 116131, 1275 Center Drive, Gainesville, FL, 32611, USA

    Gregory A. Hudalla

Authors
  1. Bryant J. Kane
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margaret M. Fettis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaheen A. Farhadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Renjie Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gregory A. Hudalla
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gregory A. Hudalla.

Additional information

Associate Editor Michael R. King oversaw the review of this article.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

Keywords

Search

Navigation