Galectin-1 is an immunomodulatory carbohydrate-binding protein with demonstrated efficacy in various preclinical models. However, its potential for clinical use is challenged by two features of the protein. First, galectin-1 (Gal-1) can be inactivated in oxidative environments, such as sites of inflammation, via covalent cross-linking of surface-exposed cysteine residues. Second, the active conformation of galectin-1 is a noncovalent homodimer with a micromolar dissociation constant. Together, these features necessitate frequent administration of high doses of galectin-1 for therapeutic efficacy. To address this challenge, we report an engineered dimeric variant of Gal-1 that is resistant to oxidative inactivation. Specifically, to prevent oxidative inactivation we mutated 3 of 4 surface cysteine residues to serine residues on Gal-1 (“Tri Gal-1”), and then cross-linked two Tri Gal-1 molecules with poly(ethylene glycol) diacrylate to create a stable homodimer (“Tri-PEG-Tri”). Our data demonstrate that cysteine-to-serine galectin-1 mutants retain the carbohydrate-binding properties and pro-apoptotic activity of wild-type Gal-1. Mutants lacking all surface cysteine residues are completely resistant to covalent cross-linking in oxidative environments. At sufficient polymer:protein ratios, poly(ethylene glycol) diacrylate reacts with the surface cysteine on two Tri Gal-1 molecules to form Tri-PEG-Tri. The effective dose of Tri-PEG-Tri is more than an order of magnitude lower than that of non-PEGylated Gal-1. Together, these data demonstrate reactive oxygen species (ROS)-resistant Tri-PEG-Tri dimers with enhanced lectin activity that may be broadly useful for improving the therapeutic efficacy of Gal-1 in immune modulation, transplant tolerance, and treatment of chronic inflammation.

中文翻译：

---

具有增强的凝集素活性的工程活性氧抗性Galectin-1二聚体

Galectin-1是一种免疫调节性碳水化合物结合蛋白，在各种临床前模型中均证明具有疗效。然而，其临床应用潜力受到蛋白质的两个特征的挑战。首先，通过表面暴露的半胱氨酸残基的共价交联，可以在氧化环境（例如炎症部位）中灭活半乳糖凝集素-1（Gal-1）。其次，galectin-1的活性构象是具有微摩尔解离常数的非共价同二聚体。在一起，这些特征需要频繁给予高剂量的半乳凝素-1以达到治疗效果。为了解决这一挑战，我们报道了一种抗氧化失活的Gal-1工程二聚体变体。具体而言，为防止氧化失活，我们将4个表面半胱氨酸残基中的3个突变为Gal-1（“ Tri Gal-1”）上的丝氨酸残基，然后将两个Tri Gal-1分子与聚（乙二醇）二丙烯酸酯交联，生成稳定的均二聚体（“ Tri-PEG-Tri”）。我们的数据表明，半胱氨酸到丝氨酸的半乳糖凝集素1突变体保留了野生型Gal-1的碳水化合物结合特性和促凋亡活性。缺少所有表面半胱氨酸残基的突变体在氧化环境中完全抵抗共价交联。在足够高的聚合物：蛋白质比率下，聚乙二醇二丙烯酸酯与两个Tri Gal-1分子上的表面半胱氨酸反应形成Tri-PEG-Tri。Tri-PEG-Tri的有效剂量比非PEG化的Gal-1的有效剂量低一个数量级以上。一起，