Inhibition of E-cadherin/catenin complex formation by O-linked N-acetylglucosamine transferase is partially independent of its catalytic activity
Inhibition of E-cadherin/catenin complex formation by O-linked N-acetylglucosamine transferase is partially independent of its catalytic activity
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O-连接 N-乙酰氨基葡萄糖转移酶对 E-钙粘蛋白/连环蛋白复合物形成的抑制部分与其催化活性无关。
DOI：
10.3892/mmr.2015.4718
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发表时间：
2016-02-01
影响因子：
3.4
通讯作者：
Yu, Wengong
中科院分区：
文献类型：
--
作者：
Liu, Hainan;Gu, Yuchao;Yu, Wengong
p120-连环蛋白（p120）含有一个大的中央犰狳重复结构域，通过它与E-钙粘蛋白结合以稳定后者，从而调节细胞与细胞的粘附。本课题组的前期研究表明，O-连接的N-乙酰葡萄糖胺（O-GlcNAc）参与调节p120和E-cadherin之间的相互作用。由于O-GlcNAc转移酶（OGT）能够直接结合其大多数靶蛋白，本研究假设OGT可能另外调节E-钙粘蛋白/连环蛋白复合物的形成，而不依赖于其催化活性。为了验证这一假设，在H1299细胞中表达催化失活的OGT突变体，并评估其对E-钙粘蛋白/连环蛋白复合物形成的影响。一个细胞粘附分子结合蛋白提取试验证实，OGT抑制的E-钙粘蛋白/连环蛋白复合物的形成独立于其催化活性。此外，免疫共沉淀和pull-down试验被用来评估OGT和p120之间的相互作用。免疫印迹表明OGT能够直接与p120结合。为了确定参与OGT结合的p120结构域，构建了一系列p120缺失突变体，并通过下拉测定进行蛋白结合测定。免疫印迹显示OGT与p120的调控区和Armadillo区结合，可能干扰p120与E-cadherin的相互作用。最后，将OGT、p120和E-cadherin胞浆区（ECD）在BL 21（DE 3）重组大肠杆菌中进行了重组表达。大肠杆菌细胞，谷胱甘肽S-转移酶（GST）下拉试验，以评估纯化的重组蛋白之间的相互作用。免疫印迹结果表明，麦芽糖结合蛋白（MBP）-OGT可抑制His-p120与GST-ECD的结合，并呈剂量依赖性。以上结果提示OGT通过降低p120与E-cadherin的相互作用抑制E-cadherin/catenin复合物的形成。本研究提供了一种新的潜在机制，调节p120和E-钙粘蛋白之间的相互作用，从而E-钙粘蛋白介导的细胞-细胞粘附，这在癌症的发展和进展中具有重要作用。
p120-catenin (p120) contains a large central armadillo repeat domain, via which it binds to E-cadherin to stabilize the latter, thereby regulating cell-to-cell adhesion. A previous study by our group demonstrated that O-linked N-acetylglucosamine (O-GlcNAc) is involved in the regulation of the interaction between p120 and E-cadherin. As O-GlcNAc transferase (OGT) is able to directly bind to the majority of its target proteins, the present study hypothesized that OGT may additionally regulate the formation of the E-cadherin/catenin complex independent of its catalytic activity. To verify this hypothesis, a catalytically inactive OGT mutant was expressed in H1299 cells, and its effects on the formation of the E-cadherin/catenin complex were assessed. A cytoskeleton-binding protein extraction assay confirmed that OGT inhibited the formation of the E-cadherin/catenin complex independent of its catalytic activity. In addition, co-immunoprecipitation and pull-down assays were used to evaluate the interaction between OGT and p120. Immunoblotting indicated that OGT was able to directly bind to p120. To determine the domain of p120 involved in binding to OGT, a series of deletion mutants of p120 were constructed and subjected to protein binding assays by pull-down assays. Immunoblotting showed that OGT bound to the regulatory and armadillo domains of p120, which might interfere with the interaction between p120 and E-cadherin. Finally, OGT, p120 and E-cadherin cytoplasmic domains (ECD) were recombinantly expressed in BL21 (DE3) recombinant E. coli cells, and a glutathione S-transferase (GST) pull-down assay was performed to assess the interactions among the purified recombinant proteins. Immunoblotting indicated that maltose-binding protein (MBP)-OGT inhibited the binding of His-p120 to GST-ECD in a dose-dependent manner. All of these results suggested that OGT inhibited the formation of the E-cadherin/catenin complex through reducing the interaction between p120 and E-cadherin. The present study provided a novel underlying mechanism of the regulation of the interaction between p120 and E-cadherin, and thus E-cadherin-mediated cell-cell adhesion, which has essential roles in cancer development and progression.