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

Lee MJ, Heo YM, Hong SH, Kim K, Park S - Immune Netw (2009)

Bottom Line: The recombinant protein was purified by protein A sepharose column chromatography.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.Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4(+)CD25(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Ajou University School of Medicine, Suwon 442-721, Korea.

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.

No MeSH data available.


The binding activity of Tim-3-Ig N-glycosylation mutants to CD4+CD25+ T cells. Mouse CD4+ T cells were labeled with FITC conjugated anti-CD4 Ab, PE-conjugated anti-CD25 Ab and either Tim-3-Ig wild type or Tim-3-Ig N-glycosylation mutants. Subsequently, these cells were incubated with biotin-conjugated anti-human IgG Ab and Streptavidin-PerCP. Cells were gated on CD4 CD25 expression and the binding activity of Tim-3-Ig to these cells was analyzed by flow cytometry.
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Figure 5: The binding activity of Tim-3-Ig N-glycosylation mutants to CD4+CD25+ T cells. Mouse CD4+ T cells were labeled with FITC conjugated anti-CD4 Ab, PE-conjugated anti-CD25 Ab and either Tim-3-Ig wild type or Tim-3-Ig N-glycosylation mutants. Subsequently, these cells were incubated with biotin-conjugated anti-human IgG Ab and Streptavidin-PerCP. Cells were gated on CD4 CD25 expression and the binding activity of Tim-3-Ig to these cells was analyzed by flow cytometry.

Mentions: Next the ligand binding activity of N-glycosylation mutant forms was examined (Fig. 5). The concentration of each Tim-3-Ig form was adjusted to 0.75 µM. Since the concentration of Tim-3-Ig was much lower than that used in Fig. 3A, the binding activity of wild Tim-3-Ig was less evident. However, as expected, there was no difference in the binding activity between any of the Tim-3-Ig N-glycosylation mutant forms and wild type Tim-3-Ig implying that N-glycosylation of Tim-3 IgV like domain may not affect ligand binding activity of Tim-3.


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

Lee MJ, Heo YM, Hong SH, Kim K, Park S - Immune Netw (2009)

The binding activity of Tim-3-Ig N-glycosylation mutants to CD4+CD25+ T cells. Mouse CD4+ T cells were labeled with FITC conjugated anti-CD4 Ab, PE-conjugated anti-CD25 Ab and either Tim-3-Ig wild type or Tim-3-Ig N-glycosylation mutants. Subsequently, these cells were incubated with biotin-conjugated anti-human IgG Ab and Streptavidin-PerCP. Cells were gated on CD4 CD25 expression and the binding activity of Tim-3-Ig to these cells was analyzed by flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803304&req=5

Figure 5: The binding activity of Tim-3-Ig N-glycosylation mutants to CD4+CD25+ T cells. Mouse CD4+ T cells were labeled with FITC conjugated anti-CD4 Ab, PE-conjugated anti-CD25 Ab and either Tim-3-Ig wild type or Tim-3-Ig N-glycosylation mutants. Subsequently, these cells were incubated with biotin-conjugated anti-human IgG Ab and Streptavidin-PerCP. Cells were gated on CD4 CD25 expression and the binding activity of Tim-3-Ig to these cells was analyzed by flow cytometry.
Mentions: Next the ligand binding activity of N-glycosylation mutant forms was examined (Fig. 5). The concentration of each Tim-3-Ig form was adjusted to 0.75 µM. Since the concentration of Tim-3-Ig was much lower than that used in Fig. 3A, the binding activity of wild Tim-3-Ig was less evident. However, as expected, there was no difference in the binding activity between any of the Tim-3-Ig N-glycosylation mutant forms and wild type Tim-3-Ig implying that N-glycosylation of Tim-3 IgV like domain may not affect ligand binding activity of Tim-3.

Bottom Line: The recombinant protein was purified by protein A sepharose column chromatography.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.Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4(+)CD25(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Ajou University School of Medicine, Suwon 442-721, Korea.

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.

No MeSH data available.