A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction

Autoři: Nobuo Watanabe aff001;  Masako Kidokoro aff001;  Yusuke Suzuki aff001;  Makiko Tanaka aff001;  Shigeaki Inoue aff001;  Hideo Tsukamoto aff002;  Noriaki Hirayama aff003;  Pei-Wen Hsieh aff004;  Ching-Ping Tseng aff006;  Yoshihide Nakagawa aff001;  Sadaki Inokuchi aff001
Působiště autorů: Department of Emergency and Critical Care Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan aff001;  Department of the Education and the Research Support Center Tokai University School of Medicine, Isehara, Kanagawa, Japan aff002;  Institute of Advanced Biosciences, Tokai University, Isehara, Kanagawa, Japan aff003;  Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China aff004;  Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan, Republic of China aff005;  Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China aff006;  Graduate Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China aff007;  Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan, Republic of China aff008
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222331


Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.

Klíčová slova:

Binding analysis – Disulfide bonds – HeLa cells – Membrane potential – Plasmid construction – Protein interactions – Platelet aggregation – Platelet activation


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