Cellular and Structural Biology Division, ICMR-National Institute for Research in Reproductive Health, Mumbai, India.
Cellular and Structural Biology Division, ICMR-National Institute for Research in Reproductive Health, Mumbai, India.
Arch Biochem Biophys. 2021 Dec 15;714:109084. doi: 10.1016/j.abb.2021.109084. Epub 2021 Nov 10.
Trophoblast antigen 2 (Trop2) is a type I transmembrane protein post-translationally modified by N-linked glycosylation. It was originally detected in trophoblasts but was later shown to be frequently overexpressed in many epithelial cancers. Recently, anti-Trop2 antibody-drug conjugate has been FDA approved for the treatment of metastatic triple-negative breast and urothelial carcinomas, making it an important tumor antigen. The current study explored the significance of N-glycosylation of Trop2 by substituting specific N-glycan addition sites by site-directed mutagenesis. The mutant proteins were characterized in transiently transfected HEK293 cells. The N-glycosylation mutants did not affect protein expression, stability, dimerization ability and matriptase mediated cleavage. However, NA and NA mutants showed decreased interaction with its binding partner claudin-7. Our earlier reported Trop2 mutant VA, which shows aberrant glycosylation, also displayed hampered interaction with claudin-7. To further characterize the mutants, stable clones expressing wild type and mutant Trop2 were generated in OVCAR3 cell line. Interestingly, surface biotinylation assay showed significantly higher surface expression of NA and NA mutants whereas surface localization was drastically reduced for VA Trop2 mutant. Though overexpression of wild type Trop2 did not cause any change in fibronectin-mediated FAK (Focal adhesion kinase) signaling; expression of NA mutant, surprisingly downregulated FAK signaling. Furthermore, exosomal release of Trop2 was also decreased in NA and NA mutants. This data suggests that site-specific N-glycan addition determines Trop2 surface density, claudin-7 interaction and exosomal release.
滋养层抗原 2（Trop2）是一种 I 型跨膜蛋白，经翻译后修饰发生 N-连接糖基化。最初在滋养层细胞中检测到，但后来发现其在许多上皮性癌中频繁过表达。最近，抗 Trop2 抗体药物偶联物已获得 FDA 批准，用于治疗转移性三阴性乳腺癌和尿路上皮癌，使其成为一种重要的肿瘤抗原。本研究通过定点突变取代特定的 N-糖基化添加位点来探讨 Trop2 的 N-糖基化意义。突变蛋白在瞬时转染的 HEK293 细胞中进行了表征。N-糖基化突变体不影响蛋白表达、稳定性、二聚化能力和基质金属蛋白酶（matriptase）介导的切割。然而，NA 和 NA 突变体与结合伴侣 Claudin-7 的相互作用减弱。我们之前报道的 Trop2 突变体 VA 显示异常糖基化，也显示与 Claudin-7 的相互作用受阻。为了进一步表征突变体，在 OVCAR3 细胞系中生成了表达野生型和突变型 Trop2 的稳定克隆。有趣的是，表面生物素化实验表明，NA 和 NA 突变体的表面表达显著增加，而 VA Trop2 突变体的表面定位则明显减少。尽管过表达野生型 Trop2 不会引起纤连蛋白介导的 FAK（黏着斑激酶）信号的任何变化；但出乎意料的是，NA 突变体的表达下调了 FAK 信号。此外，NA 和 NA 突变体中 Trop2 的外泌体释放也减少了。这些数据表明，特定位置的 N-糖基化决定了 Trop2 的表面密度、Claudin-7 的相互作用和外泌体的释放。
