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.

中文翻译：

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Galectin-1 同源二聚体通过聚（乙二醇）双马来酰亚胺的位点特异性交联

介绍

天然免疫调节剂 Galectin-1 (Gal1) 作为一种免疫调节治疗剂的前景受到其不稳定的同源二聚体构象的挑战。以前，通过共价聚 (乙二醇) 二丙烯酸酯 (PEGDA) 交联稳定的 Gal1 同源二聚体相对于非共价同源二聚体表现出更高的活性。

方法

在这里，我们报告了使用另一种硫醇-迈克尔加成接头化学形成的 Gal1 同型二聚体。

结果

聚 (乙二醇) 双马来酰亚胺 (PEGbisMal) 在多个位点与 Gal1 反应，其效率高于 PEGDA。然而，多个 PEGbisMal 分子与 Gal1 C130 结合，这是一种 Gal1 突变体，具有一个表面半胱氨酸 (cys-130) 和两个被认为埋在溶剂无法进入的蛋白质核心 (cys-42 和 cys-60) 中的半胱氨酸。定点诱变表明 cys-60 是其他 PEGbisMal 分子与 Gal1 C130 结合的位点。与 WT-Gal1 相比，Gal1 C130 诱导 Jurkat T 细胞死亡的活性较低，其特征在于磷脂酰丝氨酸暴露和膜通透性。PEG交联可以恢复Gal1 C130的功能，使得高浓度下PEGbisMal交联的Gal1 C130比PEGDA交联的WT-Gal1和Gal1 C130具有更高的活性。将 Gal1 C130 中的 cys-42 和 cys-60 突变为丝氨酸不影响由 PEGbisMal 交联的 Gal1 C130 同源二聚体的细胞死亡信号传导活性。聚乙二醇化的 Gal1 C130 变体还消除了对维持 WT-Gal1 信号传导活性所需的还原剂（例如二硫苏糖醇）的需求。

结论

总的来说，这些数据表明，硫醇-迈克尔加成生物共轭导致 PEG 交联的 Gal1 同源二聚体具有改善的细胞外信号活性，不需要还原环境即可发挥作用。