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The Binding Properties of Glycosylated and Non- Glycosylated Tim-3 Molecules on $CD4^+CD25^+$T Cells

Lee, Mi-Jin    (Department of Microbiology and Immunology, Ajou University School of Medicine   ); Heo, Yoo-Mi    (Department of Microbiology and Immunology, Ajou University School of Medicine   ); Hong, Seung-Ho    (Department of Microbiology and Immunology, Ajou University School of Medicine   ); Kim, Kyong-Min    (Department of Microbiology and Immunology, Ajou University School of Medicine   ); Park, Sun    (Department of Microbiology and Immunology, Ajou University School of Medicine  );
  • 초록

    Background: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on $CD4^+CD25^+$ T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to $CD4^+CD25^+$ T cells. Methods: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to $CD4^+CD25^+$ T cells was analyzed using flow cytometry. Results: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to $CD4^+CD25^+$ T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. Conclusion: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on $CD4^+CD25^+$ T cells.


  • 주제어

    Tim-3 .   N-glycosylation .   Tim-3L .   $CD4^+CD25^+$T cells.  

  • 참고문헌 (11)

    1. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, Manning S, Greenfield EA, Coyle AJ, Sobel RA, Freeman GJ, Kuchroo VK: Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 415;536-541, 2002 
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