Department of Immunology, Faculty of Medicine, Kindai University, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Osaka, Japan.
Department of Anesthesiology, Faculty of Medicine, Kindai University, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Osaka, Japan.
Viruses. 2022 Apr 17;14(4):832. doi: 10.3390/v14040832.
Tissue and subcellular localization and its changes upon cell activation of virus-restricting APOBEC3 at protein levels are important to understanding physiological functions of this cytidine deaminase, but have not been thoroughly analyzed in vivo. To precisely follow the possible activation-induced changes in expression levels of APOBEC3 protein in different mouse tissues and cell populations, genome editing was utilized to establish knock-in mice that express APOBEC3 protein with an in-frame FLAG tag. Flow cytometry and immunohistochemical analyses were performed prior to and after an immunological stimulation. Cultured B cells expressed higher levels of APOBEC3 protein than T cells. All differentiation and activation stages of freshly prepared B cells expressed significant levels of APOBEC3 protein, but germinal center cells possessed the highest levels of APOBEC3 protein localized in their cytoplasm. Upon immunological stimulation with sheep red blood cells in vivo, germinal center cells with high levels of APOBEC3 protein expression increased in their number, but FLAG-specific fluorescence intensity in each cell did not change. T cells, even those in germinal centers, did not express significant levels of APOBEC3 protein. Thus, mouse APOBEC3 protein is expressed at distinctively high levels in germinal center B cells. Antigenic stimulation did not affect expression levels of cellular APOBEC3 protein despite increased numbers of germinal center cells.
在蛋白质水平上，研究病毒限制因子 APOBEC3 的组织和亚细胞定位及其在细胞激活后的变化，对于理解这种胞嘧啶脱氨酶的生理功能非常重要，但在体内尚未得到彻底分析。为了准确跟踪 APOBEC3 蛋白在不同小鼠组织和细胞群中的可能激活诱导的表达水平变化，利用基因组编辑技术建立了表达带有框架内 FLAG 标签的 APOBEC3 蛋白的敲入小鼠。在免疫刺激前后进行了流式细胞术和免疫组织化学分析。培养的 B 细胞表达的 APOBEC3 蛋白水平高于 T 细胞。新鲜制备的 B 细胞的所有分化和激活阶段均表达显著水平的 APOBEC3 蛋白，但生发中心细胞具有定位于其细胞质中的最高水平的 APOBEC3 蛋白。在体内用绵羊红细胞进行免疫刺激后，高水平表达 APOBEC3 蛋白的生发中心细胞数量增加，但每个细胞中的 FLAG 特异性荧光强度没有变化。T 细胞，即使是生发中心的 T 细胞，也没有表达显著水平的 APOBEC3 蛋白。因此，小鼠 APOBEC3 蛋白在生发中心 B 细胞中表达水平明显较高。尽管生发中心细胞数量增加，但抗原刺激并未影响细胞 APOBEC3 蛋白的表达水平。
