The TNF-TNFR1 signaling pathway plays a pivotal role in regulating the balance between cell survival and cell death. Upon binding to TNF, plasma membrane-localized TNFR1 initiates survival signaling, whereas TNFR1 internalization promotes caspase-mediated apoptosis. We previously reported that the α2-6 sialylation of TNFR1 by the tumor-associated sialyltransferase ST6GAL1 diverts signaling toward survival by inhibiting TNFR1 internalization. In the current investigation, we interrogated the mechanisms underlying sialylation-dependent regulation of TNFR1 and uncovered a novel role for α2-6 sialylation, but not α2-3 sialylation, in mediating apoptosis-resistance. Our studies utilized HEK293 cells with deletion of sialyltransferases that modify N-glycans with either α2-3-linked sialic acids (ST3GAL3/4/6) or α2-6-linked sialic acids (ST6GAL1/2). Additionally, ST6GAL1 was re-expressed in cells with ST6GAL1/2 deletion to restore α2-6 sialylation. Using total internal reflection fluorescence (TIRF) microscopy and BS3 cross-linking, we determined that, under basal conditions, cells expressing TNFR1 devoid of α2-6 sialylation displayed enhanced TNFR1 oligomerization, an event that P cells for activation by TNF. Moreover, upon stimulation with TNF, greater internalization of TNFR1 was observed via time-lapse TIRF and flow cytometry, and this correlated with increased caspase-dependent apoptosis. These effects were reversed by ST6GAL1 re-expression. Conversely, eliminating α2-3 sialylation did not significantly alter TNFR1 clustering, internalization or apoptosis. We also evaluated the Fas receptor, given its structural similarity to TNFR1. As with TNFR1, α2-6 sialylation had a selective effect in protecting cells against Fas-mediated apoptosis. These results collectively suggest that ST6GAL1 may serve a unique function in shielding cancer cells from apoptotic stimuli within the tumor microenvironment.

中文翻译：

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TNFR1 的凋亡信号传导受 TNFR1 N-糖的 α2-6 唾液酸化抑制，但 α2-3 唾液酸化不受抑制


TNF-TNFR1 信号通路在调节细胞存活和细胞死亡之间的平衡中起关键作用。与 TNF 结合后，质膜定位的 TNFR1 启动存活信号传导，而 TNFR1 内化促进 caspase 介导的细胞凋亡。我们之前报道过，肿瘤相关唾液酸转移酶对 TNFR1 的 α2-6 唾液酸化ST6GAL1通过抑制 TNFR1 内化将信号转导至生存。在目前的研究中，我们询问了 TNFR1 唾液酸化依赖性调节的潜在机制，并发现了 α2-6 唾液酸化（而不是 α2-3 唾液酸化）在介导细胞凋亡耐药性中的新作用。我们的研究利用了 HEK293 细胞，唾液酸转移酶缺失了唾液酸转移酶，这些唾液酸转移酶用 α2-3-连接唾液酸 （ST3GAL3/4/6） 或 α2-6-连接唾液酸 （ST6GAL1/2） 修饰 N-聚糖。此外，ST6GAL1 在 ST6GAL1/2 缺失的细胞中重新表达以恢复 α2-6 唾液酸化。使用全内反射荧光 （TIRF） 显微镜和 BS3 交联，我们确定，在基础条件下，表达缺乏 α2-6 唾液酸化的 TNFR1 的细胞表现出增强的 TNFR1 寡聚化，这一事件是 P 细胞被 TNF 激活。此外，在 TNF 刺激后，通过延时 TIRF 和流式细胞术观察到 TNFR1 的更大内化，这与 caspase 依赖性细胞凋亡增加相关。这些影响被 ST6GAL1 重新表达逆转。相反，消除 α2-3 唾液酸化不会显着改变 TNFR1 的聚集、内化或细胞凋亡。鉴于 Fas 受体的结构与 TNFR1 相似，我们还评估了 Fas 受体。与 TNFR1 一样，α2-6 唾液酸化在保护细胞免受 Fas 介导的细胞凋亡方面具有选择性作用。 这些结果共同表明，ST6GAL1可能在保护癌细胞免受肿瘤微环境中的凋亡刺激方面发挥独特的功能。