The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4+CD25+ T Cells.
- Author:
Mi Jin LEE
1
;
Yoo Mi HEO
;
Seung Ho HONG
;
Kyongmin KIM
;
Sun PARK
Author Information
- Publication Type:Original Article
- Keywords: Tim-3; N-glycosylation; Tim-3L; CD4+CD25+ T cells
- MeSH: Animals; Bacteria; CHO Cells; Chromatography; Clone Cells; Cricetinae; DNA, Complementary; Flow Cytometry; Immunoglobulins; Ligands; Mice; Mucins; Mutagenesis, Site-Directed; Plasmids; Proteins; Sepharose; Staphylococcal Protein A; T-Lymphocytes; Th1 Cells
- From:Immune Network 2009;9(2):58-63
- CountryRepublic of Korea
- Language:English
- Abstract: 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.
