Zhang Sipan, Yang Qianqian, Liu Zhihong, Shi Shaolin
National Clinical Research Center for Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine Nanjing, China.
Department of Medicine, Icahn School of Medicine at Mount Sinai USA.
Am J Transl Res. 2020 May 15;12(5):2212-2224. eCollection 2020.
gene encodes a O-fucosyltransferase that adds fucose to the serine/threonine residue in the sequence of CXXXX(S/T)C of EGF-like domain in a protein. O-fucosylation has been shown to be required for some EGF-like domain-containing proteins to function, e.g., Notch1, and POFUT1 deficiency could affect cellular function and cause diseases. is ubiquitously expressed, but its essentiality for most cell types is not known. In the present study, we examined the consequence of Pofut1 gene abrogation in mouse podocytes using Cre-loxP system, and found that the conditional knockout mice were indistinguishable from wild-type controls in urinary protein level, glomerular morphology, podocyte foot process ultrastructure, podocyte marker expression and podocyte numbers. These results indicated that POFUT1 is not essential for podocyte structure, function and survival in mice. To understand why POFUT1 is dispensable for podocytes, we searched mouse podocyte essential gene candidates (as determined by single-cell RNA-seq) and found only two POFUT1 substrates, NOTCH2 and tPA. It has been shown that abrogation of these genes does not cause podocyte injury, explaining dispensability of POFUT1 for mouse podocytes and demonstrating a feasibility to predict POFUT1 essentiality for a given cell type. At present, most mouse cell types have been subject to single-cell RNA-seq, making essential gene prediction and thus POFUT1 requirement prediction possible for the cell types.
基因编码一种O-岩藻糖基转移酶，该酶可将岩藻糖添加到蛋白质中表皮生长因子（EGF）样结构域的CXXXX(S/T)C序列中的丝氨酸/苏氨酸残基上。已证明O-岩藻糖基化对于某些含EGF样结构域的蛋白质发挥功能是必需的，例如Notch1，而POFUT1缺陷可能影响细胞功能并导致疾病。POFUT1在全身广泛表达，但其对大多数细胞类型的必要性尚不清楚。在本研究中，我们使用Cre-loxP系统检测了小鼠足细胞中Pofut1基因缺失的后果，发现条件性敲除小鼠在尿蛋白水平、肾小球形态、足细胞足突超微结构、足细胞标志物表达和足细胞数量方面与野生型对照无差异。这些结果表明，POFUT1对小鼠足细胞的结构、功能和存活并非必需。为了理解为什么POFUT1对足细胞是可有可无的，我们搜索了小鼠足细胞必需基因候选物（由单细胞RNA测序确定），仅发现两个POFUT1底物，NOTCH2和组织型纤溶酶原激活剂（tPA）。已表明这些基因的缺失不会导致足细胞损伤，这解释了POFUT1对小鼠足细胞的可有可无性，并证明了预测POFUT1对特定细胞类型必要性的可行性。目前，大多数小鼠细胞类型都已进行单细胞RNA测序，使得对这些细胞类型进行必需基因预测以及因此预测POFUT1的需求成为可能。
