Inhibition of E-cadherin/catenin complex formation by O-linked N-acetylglucosamine transferase is partially independent of its catalytic activity

Liu,Haiyan; Gu,Yuchao; Qi,Jieqiong; Han,Cuifang; Zhang,Xinling; Bi,Chuanlin; Yu,Wengong

doi:10.3892/mmr.2015.4718

Inhibition of E-cadherin/catenin complex formation by O-linked N-acetylglucosamine transferase is partially independent of its catalytic activity

Authors:
- Haiyan Liu
- Yuchao Gu
- Jieqiong Qi
- Cuifang Han
- Xinling Zhang
- Chuanlin Bi
- Wengong Yu
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Affiliations: Department of Glycobiology, School of Medicine and Pharmacy, Ocean University of China, Qingdao, Shandong 266003, P.R. China
Published online on: December 24, 2015 https://doi.org/10.3892/mmr.2015.4718
Pages: 1851-1860

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Abstract

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.

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