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    刘宇博

    • 教授     博士生导师   硕士生导师
    • 任职 : 智能生物制造教育部重点实验室
    • 性别:男
    • 毕业院校:大连理工大学
    • 学位:博士
    • 所在单位:化工海洋与生命学院
    • 学科:生物化学与分子生物学. 生物化工. 化学生物学
    • 办公地点:大连理工大学 智能生物制造教育部重点实验室 生命科学与药学系
    • 联系方式:liuyubo@dlut.edu.cn
    • 电子邮箱:liuyubo@dlut.edu.cn

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

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    论文类型:期刊论文

    发表时间:2019-06-18

    发表刊物:BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

    收录刊物:PubMed、SCIE

    卷号:514

    期号:1

    页面范围:157-165

    ISSN号:0006-291X

    关键字:O-GalNAcylation; FOXA1; ppGalNAc-T2; In vitro glycosylation

    摘要: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 ER alpha 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 ER alpha 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, 5354 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 5355 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. (C) 2019 Elsevier Inc. All rights reserved.