title: 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.
  
  
  CONCLUSIONS
  Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4(+)CD25(+) T cells.
authors.affiliation: Department of Microbiology and Immunology, Ajou University School of Medicine, Suwon 442-721, Korea.
authors.forename: Mi Jin
authors.initials: MJ
authors.lastname: Lee
authors.forename: Yoo Mi
authors.initials: YM
authors.lastname: Heo
authors.forename: Seung-Ho
authors.initials: SH
authors.lastname: Hong
authors.forename: Kyongmin
authors.initials: K
authors.lastname: Kim
authors.forename: Sun
authors.initials: S
authors.lastname: Park