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The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4CD25 T Cells.

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.

SUBMITTER: Lee MJ

PROVIDER: S-EPMC2803304 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4CD25 T Cells.

Lee Mi Jin MJ Heo Yoo Mi YM Hong Seung-Ho SH Kim Kyongmin K Park Sun S

Immune network 20090430 2

<h4>Background</h4>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 protein ...[more]

PMID: 20107545

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Project description:BackgroundT-cell immunoglobulin and mucin domain (Tim) proteins are immunomodulatory molecules that play key roles in the regulation of T-cell activation. Published studies have reported that Tim molecules are involved in the pathogenesis of certain autoimmune diseases. Type 1 diabetes (T1D) is an autoimmune disease in which T cells mediate the destruction of islet β cells. However, the expression of Tim molecules in T1D remains unclear. In this study, we measured the expression of Tim family molecules as well as T-cell subset-specific transcription factors in T1D patients, and we explored the possible involvement of Tim molecules in the pathogenesis of T1D.MethodsNinety T1D patients, Thirty-six type 2 diabetes (T2D) patients and forty healthy controls (HCs) were recruited for this study. Peripheral blood mononuclear cells (PBMCs) were isolated, RNA was extracted from the PBMCs and reverse transcribed into cDNA, and gene expression patterns were analysed by RT-qPCR. The expression of Tim molecules in different T-cell subsets was analysed by flow cytometry.ResultsCompared with that in HCs, the mRNA expression of Tim-1 and RORC was increased in T1D patients (P=0.0355 and P=0.0423, respectively), while the expression of Tim-3 was decreased (P=0.0013). In addition, compared with HCs, the ratio of Tim-3 to Tim-1 expression in diabetic patients was decreased (P<0.0001 for T1D and P=0.0387 for T2D). The ratios of T-Bet to GATA3 expression and RORC to FOXP3 expression were higher in T1D patients than in HCs (P=0.0042 and P=0.0066, respectively). Furthermore, the T1D patients with defective islet function had more significant imbalances in the Tim-3/Tim-1 and RORC/FOXP3 ratios (P<0.0001, and P=0.001, respectively). Moreover, Both Tim-3 expression in CD4+ T cells and the Tim-3 to Tim-1 ratio were elevated in T1D in the remission phase compared to T1D.ConclusionOur study revealed altered expression of Tim molecules in T1D patients. The imbalanced ratios of Tim-3/Tim-1 expression were more pronounced in T1D patients with defective islet function. However, alterations in Tim molecule expression are mitigated in T1D in the remission phase. All these findings suggest that Tim family molecules may be involved in the pathogenesis of T1D.

Dataset Information

The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4CD25 T Cells.

Publications

The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4CD25 T Cells.

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