College of Life Sciences, Wuhan University, Wuhan 430072, China.
School of Basic Medical Sciences, Xianning Medical College, Hubei University of Science and Technology, Xianning 437100, China.
Molecules. 2023 Feb 24;28(5):2129. doi: 10.3390/molecules28052129.
O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in . OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in . Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.
O-GlcNAc 修饰是由 OGT 介导的 GlcNAc 的单一糖基化，它调节底物蛋白的功能，与许多疾病密切相关。然而，大量的 O-GlcNAc 修饰靶蛋白成本高、效率低且制备复杂。在这项研究中，成功地在. 中建立了一种 OGT 结合肽（OBP）标记策略，以提高 O-GlcNAc 修饰的比例。OBP（P1、P2 或 P3）与靶蛋白 Tau 融合作为标记 Tau。Tau 或标记 Tau 与 OGT 一起共构建到. 中表达的载体中。与 Tau 相比，P1Tau 和 TauP1 的 O-GlcNAc 水平增加了 4~6 倍。此外，P1Tau 和 TauP1 提高了 O-GlcNAc 修饰的均一性。P1Tau 上的高 O-GlcNAcylation 导致其在体外的聚集速率明显慢于 Tau。该策略还成功地用于提高 c-Myc 和 H2B 的 O-GlcNAc 水平。这些结果表明，OBP 标记策略是一种成功的提高靶蛋白 O-GlcNAc 化水平的方法，可用于进一步的功能研究。
