Department of Microbiology, Blavatnik Institute of Harvard Medical School, Boston, MA 02115.
Department of Chemistry, St. Olaf College, Northfield, MN 55057.
Proc Natl Acad Sci U S A. 2021 Jan 26;118(4). doi: 10.1073/pnas.2016778118.
O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT's myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.
O-连接的 N-乙酰氨基葡萄糖转移酶（OGT）存在于所有哺乳动物细胞类型的核和细胞质中，对细胞增殖至关重要。OGT 为何对细胞生长是必需的尚不清楚。OGT 在同一个活性位点上执行两种酶促反应。一种是糖基化数千种不同的蛋白质，另一种是蛋白水解切割另一种参与基因表达的必需蛋白质。OGT 的众多细胞功能的去卷积一直具有挑战性，因为基因缺失是致命的；尚未建立互补方法。在这里，我们开发了用分离功能变体替代内源性 OGT 的方法来研究 OGT 的酶活性对细胞活力的重要性。通过遗传互补，我们发现 OGT 的糖基转移酶功能是细胞生长所必需的，但它的蛋白酶功能是可有可无的。接下来，我们使用互补方法构建了带有去稳定标签的野生型 OGT 的细胞系。当 OGT 被降解到非常低的水平时，细胞停止增殖但仍保持存活。添加无催化活性的 OGT 可恢复生长。因此，OGT 具有必需的非催化作用，这对于细胞增殖是必要的。通过开发一种方法来量化 OGT 的催化和非催化活性如何影响蛋白质丰度，我们发现 OGT 的非催化功能通常会影响与其催化功能不同的蛋白质。参与氧化磷酸化和肌动蛋白细胞骨架的蛋白质尤其受到非催化功能的影响。我们得出结论，OGT 整合了催化和非催化功能来控制细胞生理学。
