Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3–mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3–mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid–liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding–triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT–NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT–CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.

Galectin-3 (Gal-3) 具有长的、非周期性的、动态的富含脯氨酸的 N 端尾 (NT)。自发现以来，NT 及其众多脯氨酸的功能作用一直是个谜。为了解决这个难题，我们分别对前 68 个残基上的所有 14 个 NT 脯氨酸进行了突变，并评估了它们对各种 Gal-3 介导的功能的影响。我们的研究结果表明，任何单个脯氨酸（尤其是 P37A、P55A、P60A、P64A/H 和 P67A）的突变都会显着且不同地抑制 Gal-3 介导的细胞活动（即细胞迁移、活化、内吞作用和血凝作用）。对于机理洞察，我们研究了脯氨酸在介导 Gal-3 寡聚化中的作用，这是这些细胞活动所需的基本过程。我们表明，通过与糖蛋白结合引发的 Gal-3 寡聚化是一个类似于液-液相分离 (LLPS) 的动态过程。这些异源寡聚体的组成取决于 Gal-3 的浓度以及糖蛋白的浓度和类型。在质膜和破裂的内膜上也观察到 LLPS 样 Gal-3 寡聚化/缩合。基于分子和细胞的测定表明，聚糖结合触发的 Gal-3 LLPS（或 LLPS 样）主要由 Gal-3 NT 和碳水化合物识别域 (CRD) F 面之间的动态分子间相互作用驱动，尽管 NT –NT 相互作用似乎贡献较小。NT 内每个脯氨酸的突变对 NT-CRD 相互作用有不同的控制，从而影响聚糖结合、LLPS 和细胞活性。