The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4+CD25+ T Cells
Immune Network
;
: 58-63, 2009.
Article
Dans Anglais
| WPRIM
| ID: wpr-49348
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.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Plasmides
/
Agarose
/
Protéine A staphylococcique
/
Bactéries
/
Immunoglobulines
/
Lymphocytes T
/
Protéines
/
Chromatographie
/
Mutagenèse dirigée
/
Cellules CHO
Limites du sujet:
Animaux
langue:
Anglais
Texte intégral:
Immune Network
Année:
2009
Type:
Article
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