GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex post-translational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial-mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-β) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knock-out decreased O-GalNAc modification of TGF-β type Ⅰ and Ⅱ receptor (TβR Ⅰ and Ⅱ) and led to the elevation of TGF-β receptor dimerization and activity. Importantly, a peptide from TβR Ⅱ was first identified as the naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TβR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31A mutation enhanced TGF-β signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-β receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment.

中文翻译：

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ppGalNAc-T4催化TGF-βⅡ型受体O-糖基化调节乳腺癌细胞转移


GalNAc 型 O-糖基化最初由多肽 N-乙酰半乳糖胺基转移酶 (ppGalNAc-Ts) 催化，是最丰富、最复杂的蛋白质翻译后修饰之一。新的证据证明异常的 ppGalNAc-T 参与恶性肿瘤转化。然而，ppGalNAc-Ts 的确切分子功能仍不清楚。在此，研究了一种亚型 ppGalNAc-T4 在乳腺癌细胞系中的作用。发现 ppGalNAc-T4 的表达与乳腺癌细胞的迁移呈负相关。功能丧失研究表明，ppGalNAc-T4 通过抑制上皮间质转化 (EMT) 过程来减弱乳腺癌细胞的迁移和侵袭。相应地，转化生长因子β (TGF-β) 信号传导（EMT 的上游途径）受到 ppGalNAc-T4 表达的损害。 ppGalNAc-T4敲除减少了O-GalNAc对TGF-βⅠ型和Ⅱ型受体（TβRⅠ和Ⅱ）的修饰，导致TGF-β受体二聚化和活性升高。重要的是，来自TβR Ⅱ的肽首次被鉴定为ppGalNAc-T4的裸露肽底物，其亲和力高于ppGalNAc-T2。此外，对应于TβR Ⅱ胞外结构域的Ser31在体外被ppGalNAc-T4糖基化后被鉴定为O-GalNAc化位点。 O-GalNAc 缺陷的 S31A 突变增强了乳腺癌细胞中的 TGF-β 信号活性和 EMT。总之，这些结果确定了 ppGalNAc-T4 催化 TGF-β 受体 O-GalNAcylation 的新机制，可通过 EMT 过程抑制乳腺癌细胞迁移和侵袭。靶向 ppGalNAc-T4 可能是乳腺癌治疗的潜在治疗策略。