Identification of the O-GalNAcylation site(s) on FOXA1 catalyzed by ppGalNAc-T2 enzyme in vitro.

FOXA1 functions as a pioneer factor of transcriptional regulation that binds to specific sites in the chromatin and recruits other transcription factors, promoting the initiation of gene transcription and mediating the regulation of downstream target gene expression. FOXA1 was reported to facilitate or reprogram ERα binding, thus playing a key function in breast cancer progression. Our previous results indicated that the O-linked N-acetylgalactosamine (O-GalNAc) modification of FOXA1 plays a potentially significant role in the ERα transcription network. However, further investigations are needed to identify the specific mechanism of modification and the specific glycosylation sites on FOXA1. In this study, we first suggested that FOXA1 could be O-GalNAcylated by ppGalNAc-T2 in vitro. By dividing and expressing recombinant FOXA1 as three segments, two O-GalNAcylation sites were found on FOXA1, both located at the C-terminal of the protein. Then, synthesized peptides, including the predicted O-GalNAc sites in the C-terminus of FOXA1, were used in a vitro reaction, and peptides mutated at the predicted O-GalNAc sites were employed as controls. Through an ESI-MS assay, S354 and S355 were identified as probable O-GalNAcylation sites on FOXA1. Additionally, we performed ESI-ETD-MS/MS analysis of the full-length O-GalNAcylated FOXA1 protein and identified S355 as the O-GalNAc modification site on FOXA1, consistent with the peptide reaction. In conclusion, our results demonstrated that FOXA1 can be O-GalNAcylated by ppGalNAc-T2 at S355 in vitro. These results will provide new insights for studying the role of O-GalNAcylation in the development of breast cancer. • Nuclear transcription factor FOXA1, which plays a key function in tumor progression was confirmed to be O-GalNAcylated by ppGalNAc-T2 in vitro. • By dividing and expressing recombinant FOXA1 as three segments, two O-GalNAcylation sites were found on FOXA1 by HPLC and mass ESI-MS, both located at the C-terminal of the protein. • ESI-ETD-MS/MS analysis of the full-length in vitro O-GalNAcylated FOXA1 protein identified S355 as the O-GalNAc modification site on FOXA1. [ABSTRACT FROM AUTHOR]