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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.


Production of Tim-3-Ig N-glycosylation mutants. (A) Schematic representation and sequence analysis of Tim-3-Ig N-glycosylation mutants, N53Q and N100Q. (B) Tim-3-Ig N-glycosylation mutants were purified from CHO cell culture using affinity column chromatography and subjected to SDS-PAGE analysis and Western blot using peroxidase conjugated anti-human Ig antibody and ECL detection system.
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Figure 4: Production of Tim-3-Ig N-glycosylation mutants. (A) Schematic representation and sequence analysis of Tim-3-Ig N-glycosylation mutants, N53Q and N100Q. (B) Tim-3-Ig N-glycosylation mutants were purified from CHO cell culture using affinity column chromatography and subjected to SDS-PAGE analysis and Western blot using peroxidase conjugated anti-human Ig antibody and ECL detection system.

Mentions: The mucin domain of Tim-3 is not involved in ligand binding activity (2,5). N-glycosylation consensus sequence, Asn-Xaa-Ser/Thr, occurrs twice in Tim-3 IgV like domain. To further confirm the role of Tim-3 glycosylation in ligand binding activity, mutants of two potential N-glycosylation sites of Tim-3 IgV like domain were produced. Nucleotide sequences of eukaryotic expression vectors for N53Q, N100Q, and double mutant N53/100Q were confirmed respectively (Fig. 4A). Then, these mutant forms expressed in CHO cells were purified and confirmed by SDS-PAGE and Western blot (Fig. 4B). Compared with wild type Tim-3-Ig, the molecular weight of the double mutant N53/100Q was significantly smaller. These results indirectly showed the absence of N-glycosylation on these mutants.


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)

Production of Tim-3-Ig N-glycosylation mutants. (A) Schematic representation and sequence analysis of Tim-3-Ig N-glycosylation mutants, N53Q and N100Q. (B) Tim-3-Ig N-glycosylation mutants were purified from CHO cell culture using affinity column chromatography and subjected to SDS-PAGE analysis and Western blot using peroxidase conjugated anti-human Ig antibody and ECL detection system.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Production of Tim-3-Ig N-glycosylation mutants. (A) Schematic representation and sequence analysis of Tim-3-Ig N-glycosylation mutants, N53Q and N100Q. (B) Tim-3-Ig N-glycosylation mutants were purified from CHO cell culture using affinity column chromatography and subjected to SDS-PAGE analysis and Western blot using peroxidase conjugated anti-human Ig antibody and ECL detection system.
Mentions: The mucin domain of Tim-3 is not involved in ligand binding activity (2,5). N-glycosylation consensus sequence, Asn-Xaa-Ser/Thr, occurrs twice in Tim-3 IgV like domain. To further confirm the role of Tim-3 glycosylation in ligand binding activity, mutants of two potential N-glycosylation sites of Tim-3 IgV like domain were produced. Nucleotide sequences of eukaryotic expression vectors for N53Q, N100Q, and double mutant N53/100Q were confirmed respectively (Fig. 4A). Then, these mutant forms expressed in CHO cells were purified and confirmed by SDS-PAGE and Western blot (Fig. 4B). Compared with wild type Tim-3-Ig, the molecular weight of the double mutant N53/100Q was significantly smaller. These results indirectly showed the absence of N-glycosylation on these mutants.

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.