在内皮细胞中，磷脂酶C (PLC) β1 激活的 Ca2+ 是控制血管生成信号通路的关键第二信使。PLCβ1 通过与称为转枝结合因子 X (TRAX) 的细胞内蛋白结合而失活。本研究证明了 Globo H 鞘氨醇脂 (GHCer) 与 TRAX 之间的特异性相互作用，从而突出通过 PLCβ1 激活控制血管生成的新方法。采用酶联免疫吸附法 (ELISA) 和 Biacore 分析 Globo 系列糖脂 (GSL)，包括 GHCer 和分期胚胎抗原-3 鞘氨醇脂 (SSEA3Cer)，它们与 TRAX 的结合。评估人脐静脉内皮细胞 (HUVECs) 中 GSL 的血管生成作用。采用分子动力学 (MD) 模拟研究 GSL 的构象及其与 TRAX 的分子相互作用。采用激光共聚焦显微镜的荧光共振能量转移 (FRET) 分析 HUVECs，并用于验证从 TRAX 中释放出的 PLCβ1。此外，通过在小鼠的皮下植入 Matrigel 塞子进行体内实验确认了含有 GHCer 的胞外囊泡 (EVs) 的血管生成活性。 ELISA 和 Biacore 分析的结果显示，重组 TRAX 与合成 GHCer 之间的稳定复合物的 KD 值为 40.9 nM。相反，缺少 GHCer 终端富糖残基的 SSEA3Cer 的结合亲和力降低了约 1000 倍。这些结果与它们在 HUVECs 中的血管生成作用一致。MD 模拟表明，TRAX 与 GHCer 的糖基部分在氨基酸 Q223、Q219、L142、S141 和 E216 处相互作用。在平衡时，稳定的复合物维持了 4.6 ± 1.3 个氢键。Q223A 和 Q219A 双重突变的 TRAX 在 MD 模拟和 Biacore 实验中失去了与 GHCer 相互作用的能力。将 GHCer 终端富糖残基去除成为 SSEA3Cer，进而导致在 MD 模拟中 H-键数目减少到 1.2 ± 1.0。这种特定的氢键是由富糖残基的存在或缺失而影响整个糖基构象的构象变化所导致的。此外，ELISA、Biacore 和细胞内 FRET 实验证实了 GHCer 与 PLCβ1 之间竞争 TRAX 结合的情况。此外，Matrigel 塞子实验表明，在含有肿瘤分泌的 EVs 或合成 GHCer 的塞子中，血管形成非常强，但在含有 SSEA3Cer 的塞子中则没有。FRET 分析还表明，来自 EVs 的 GHCer 扰乱了 TRAX 和 PLCβ1 在细胞中的共定位。 总的来说， GHCer 中的富糖残基决定了其与 TRAX 结合的糖基构象，从而释放 TRAX 隔离的 PLCβ1，在内皮细胞中引起 Ca2+ 动员，增强了肿瘤微环境中的血管生成。©2022。作者。

In endothelial cells, phospholipase C (PLC) β1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation.Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice.The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with KD of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs.Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.© 2022. The Author(s).