Wu Tiangui, Sun Yuhan, Wang Dan, Isaji Tomoya, Fukuda Tomohiko, Suzuki Chiharu, Hanamatsu Hisatoshi, Nishikaze Takashi, Tsumoto Hiroki, Miura Yuri, Furukawa Jun-Ichi, Gu Jianguo
Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, Japan.
Division of Glyco-Systems Biology, Institute for Glyco-Core Research, Tokai National Higher Education and Research System, Nagoya, Japan.
J Biol Chem. 2024 Dec;300(12):108010. doi: 10.1016/j.jbc.2024.108010. Epub 2024 Nov 19.
Differentiation therapy is an alternative strategy used in treating chronic myelogenous leukemia to induce the differentiation of immature or cancerous cells toward mature cells and inhibit tumor cell proliferation. We aimed to explore N-glycans' roles in erythroid differentiation using the sodium butyrate (NaBu)-induced model of K562 cells (WT/NaBu cells). Here, using lectin blot, flow cytometry, real-time PCR, and mass spectrometry analyses, we demonstrated that the mRNA levels of N-acetylglucosaminyltransferase Ⅲ ((encoded by the MGAT3 gene) and its product (bisected N-glycans) were significantly increased during erythroid differentiation. To address the importance of GnTN-acetylglucosaminyltransferase-Ⅲ in this progress, we established a stable MGAT3 KO K562 cell line using the CRISPR/Cas9 technology. Compared to WT/NaBu cells, MGAT3 KO significantly impeded the progression of erythroid differentiation, as shown in decreased cell color and levels of erythroid markers, glycophorin A (CD235a), and β-globin. Consistently, MGAT3 KO mitigated the inhibitory impact of NaBu on cell proliferation. During induction, MGAT3 KO suppressed the cellular phosphorylated tyrosine and phospho-extracellular signal-regulated kinase (ERK)1/2 levels. Inhibition of the ERK/mitogen-activated protein kinase signaling pathway using U0126 blocked erythroid differentiation while concurrently suppressing the expression levels of MGAT3 and bisected N-glycans. Furthermore, the lack of bisecting GlcNAc modification on c-Kit and transferrin receptor 1 (CD71) suppressed cellular signaling and accelerated the degradation of the CD71 protein, respectively. Our study highlights the critical role of MGAT3 in regulating erythroid differentiation associated with the ERK/mitogen-activated protein kinase signaling pathway, which may shed light on identifying new differentiation therapy in chronic myelogenous leukemia.
分化疗法是治疗慢性粒细胞白血病的一种替代策略，用于诱导未成熟或癌细胞向成熟细胞分化，并抑制肿瘤细胞增殖。我们旨在利用丁酸钠（NaBu）诱导的K562细胞模型（WT/NaBu细胞）探索N-聚糖在红细胞分化中的作用。在此，我们通过凝集素印迹、流式细胞术、实时PCR和质谱分析表明，N-乙酰葡糖胺基转移酶Ⅲ（由MGAT3基因编码）及其产物（平分型N-聚糖）的mRNA水平在红细胞分化过程中显著增加。为了阐明GnT-N-乙酰葡糖胺基转移酶-Ⅲ在这一过程中的重要性，我们利用CRISPR/Cas9技术建立了稳定的MGAT3基因敲除K562细胞系。与WT/NaBu细胞相比，MGAT3基因敲除显著阻碍了红细胞分化进程，表现为细胞颜色变浅以及红细胞标志物血型糖蛋白A（CD235a）和β-珠蛋白水平降低。同样，MGAT3基因敲除减轻了NaBu对细胞增殖的抑制作用。在诱导过程中，MGAT3基因敲除抑制了细胞磷酸化酪氨酸和磷酸化细胞外信号调节激酶（ERK）1/2水平。使用U0126抑制ERK/丝裂原活化蛋白激酶信号通路可阻断红细胞分化，同时抑制MGAT3和平分型N-聚糖的表达水平。此外，c-Kit和转铁蛋白受体1（CD71）上缺乏平分型GlcNAc修饰分别抑制了细胞信号传导并加速了CD71蛋白的降解。我们的研究强调了MGAT3在调节与ERK/丝裂原活化蛋白激酶信号通路相关的红细胞分化中的关键作用，这可能为慢性粒细胞白血病新分化疗法的识别提供线索。
