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

Autor: Kane BJ; Department of Chemistry, University of Florida, Gainesville, FL 32611 USA., Fettis MM; J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611 USA., Farhadi SA; J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611 USA., Liu R; J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611 USA., Hudalla GA; J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611 USA.; Biomedical Sciences J293, PO BOX 116131, 1275 Center Drive, Gainesville, FL 32611 USA.
Jazyk: angličtina
Zdroj: Cellular and molecular bioengineering [Cell Mol Bioeng] 2021 Jun 04; Vol. 14 (5), pp. 523-534. Date of Electronic Publication: 2021 Jun 04 (Print Publication: 2021).
DOI: 10.1007/s12195-021-00681-0
Abstrakt: 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.
(© Biomedical Engineering Society 2021.)
Databáze: MEDLINE
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