Immune Netw.  2009 Apr;9(2):58-63. 10.4110/in.2009.9.2.58.

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

• Lee MJ
• Heo YM
• Hong SH
• Kim K
• Park S

• KMID: 1474596
• DOI: http://doi.org/10.4110/in.2009.9.2.58

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.

Keyword

Tim-3; N-glycosylation; Tim-3L; CD4+CD25+ T cells

MeSH Terms

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
DNA, Complementary
Immunoglobulins
Ligands
Mucins
Proteins
Sepharose
Staphylococcal Protein A

Figure

• 

  Figure 1 Comparison of Tim-3 aminoacid sequences of BALB/c with three different mouse strains. cDNA of Tim-3 was cloned from activated splenocytes of BALB/c mouse, nucleotide sequences were analyzed and deduced amino acid sequences were shown. Accession numbers of BALB/c Tim-3 are AY553334.1 for nucleotide sequences and AAS5931.1 for amino acid sequences. Accession numbers of C57BL/6, DBA/2 and AKR Tim-3 are BAE28574.1, AAL35776.1 and AAL65156.1, respectively.

• 

  Figure 2 Analysis of Tim-3-Ig purified from CHO cell and E. coli culture. (A) Tim-3-Ig was purified from culture supernatant of CHO cells transfected with pIRES2-EGFP-Tim-3-Ig using protein A affinity column chromatography and subjected to SDS-PAGE and Western blot using peroxidase conjugated anti-human IgG antibody and ECL detection system. (B) Tim-3-Ig was purified from culture supernatant of E. coli transformed with pIg20-Tim-3-Ig and subjected to SDS-PAGE and Western blot using peroxidase conjugated anti-human IgG antibody and ECL detection system.

• 

  Figure 3 The binding activity of Tim-3-Ig to CD4+CD25+ T cells. Mouse CD4+ T cells were labeled with FITC conjugated anti-CD4 Ab, PE-conjugated anti-CD25 Ab and Tim-3-Ig purified either from CHO cells (A) or from E. coli (B). Subsequently, cells were stained 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.

• 

  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.

• 

  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.

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