Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3–glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits (“Dimer,” “Trimer,” “Tetramer,” “Pentamer,” “Hexamer”) demonstrated glycan-binding properties and cell death–inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

Galectin-3 (Gal3) 表现出动态寡聚化和混杂结合，可导致协同、拮抗或非合作信号通路的伴随激活，从而改变细胞行为。通过 Gal3-聚糖结合界面的突变赋予信号通路选择性受到许多膜糖蛋白上常见碳水化合物类型的丰富性的挑战。在这里，我们使用 alpha 螺旋卷曲螺旋作为支架来创建具有定义化合价的合成 Gal3 结构，证明寡聚化可以物理调节 Gal3 的细胞外信号活动。具有 2 至 6 个 Gal3 亚基（“二聚体”、“三聚体”、“四聚体”、“五聚体”、“六聚体”）的构建物展示了聚糖结合特性和诱导细胞死亡的效力随化合价而变化。二聚体是最低功能价。与发出细胞凋亡信号并介导凝集的野生型 Gal3 不同，合成 Gal3 构建体在没有凝集的情况下诱导细胞死亡。在存在 CD45 的情况下，六聚体分布在细胞膜上，而在没有 CD45 的情况下，它通过 CD7 上发现的膜聚糖以外的膜聚糖聚集。野生型 Gal3、五聚体和六聚体需要 CD45 和 CD7 来发出细胞凋亡信号，并且在没有 CD45 的情况下，caspases 在细胞凋亡信号中的参与增加。然而，野生型 Gal3 比六聚体更依赖半胱天冬酶来发出细胞凋亡信号，而六聚体比五聚体具有更大的半胱天冬酶依赖性。六聚体信号下游半胱天冬酶活化减少导致 pannexin-1 半通道开放和白细胞介素 2 分泌减少，野生型 Gal3 信号通路下游半胱天冬酶活化增加促进的事件。因此，Gal3 多价性的合成固定可以通过控制膜糖蛋白的结合，进而控制细胞内通路的激活，从而对其由外而内的信号活性进行物理控制。