N-glycosylation status of Trop2 impacts its surface density, interaction with claudin-7 and exosomal release

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, N¹²⁰A and N²⁰⁸A mutants showed decreased interaction with its binding partner claudin-7. Our earlier reported Trop2 mutant V¹⁹⁴A, 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 N¹²⁰A and N²⁰⁸A mutants whereas surface localization was drastically reduced for V¹⁹⁴A Trop2 mutant. Though overexpression of wild type Trop2 did not cause any change in fibronectin-mediated FAK (Focal adhesion kinase) signaling; expression of N¹²⁰A mutant, surprisingly downregulated FAK signaling. Furthermore, exosomal release of Trop2 was also decreased in N¹²⁰A and N²⁰⁸A mutants. This data suggests that site-specific N-glycan addition determines Trop2 surface density, claudin-7 interaction and exosomal release.