Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide
Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide
复制标题
DOI：
10.1007/s12195-021-00681-0
复制
发表时间：
2021-06-04
影响因子：
2.8
通讯作者：
Hudalla, Gregory A.
中科院分区：
文献类型：
--
作者：
Kane, Bryant J.;Fettis, Margaret M.;Hudalla, Gregory A.
简介 天然免疫调节剂 Galectin-1 (Gal1) 作为免疫调节治疗剂的前景因其不稳定的同二聚体构象而受到挑战。此前，通过共价聚乙二醇二丙烯酸酯 (PEGDA) 交联稳定的 Gal1 同二聚体表现出相对于非共价同二聚体更高的活性。方法在这里，我们报告了使用替代的硫醇-迈克尔加成连接化学形成的 Gal1 同二聚体。结果 聚乙二醇双马来酰亚胺 (PEGbisMal) 与 Gal1 在多个位点发生反应，效率高于 PEGDA。然而，多个 PEGbisMal 分子与 Gal1 C130 缀合，Gal1 C130 是一种 Gal1 突变体，具有一个表面半胱氨酸 (cys-130) 和两个被认为埋藏在溶剂不可接近的蛋白质核心中的半胱氨酸 (cys-42 和 cys-60)。定点诱变证明 cys-60 是额外的 PEGbisMal 分子缀合到 Gal1 C130 上的位点。与 WT-Gal1 相比，Gal1 C130 诱导 Jurkat T 细胞死亡的活性较低，其特征是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，使得在高浓度下，PEGbisMal交联的Gal1 C130比WT-Gal1和PEGDA交联的Gal1 C130具有更高的活性。将 Gal1 C130 中的 cys-42 和 cys-60 突变为丝氨酸不会影响 PEGbisMal 交联的 Gal1 C130 同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 同二聚体具有改善的细胞外信号传导活性，并且不需要还原环境即可发挥作用。
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.