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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. CONCLUSION:Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4(+)CD25(+) T cells.

SUBMITTER: Lee MJ

PROVIDER: S-EPMC2803304 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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

Lee Mi Jin MJ Heo Yoo Mi YM Hong Seung-Ho SH Kim Kyongmin K Park Sun S

Immune network 20090430 2

<h4>Background</h4>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 protein ...[more]

PMID: 20107545

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Project description:BackgroundHuman triosephosphate isomerase (HsTIM) deficiency is a genetic disease caused often by the pathogenic mutation E104D. This mutation, located at the side of an abnormally large cluster of water in the inter-subunit interface, reduces the thermostability of the enzyme. Why and how these water molecules are directly related to the excessive thermolability of the mutant have not been investigated in structural biology.ResultsThis work compares the structure of the E104D mutant with its wild type counterparts. It is found that the water topology in the dimer interface of HsTIM is atypical, having a "wet-core-dry-rim" distribution with 16 water molecules tightly packed in a small deep region surrounded by 22 residues including GLU104. These water molecules are co-conserved with their surrounding residues in non-archaeal TIMs (dimers) but not conserved across archaeal TIMs (tetramers), indicating their importance in preserving the overall quaternary structure. As the structural permutation induced by the mutation is not significant, we hypothesize that the excessive thermolability of the E104D mutant is attributed to the easy propagation of atoms' flexibility from the surface into the core via the large cluster of water. It is indeed found that the B factor increment in the wet region is higher than other regions, and, more importantly, the B factor increment in the wet region is maintained in the deeply buried core. Molecular dynamics simulations revealed that for the mutant structure at normal temperature, a clear increase of the root-mean-square deviation is observed for the wet region contacting with the large cluster of interfacial water. Such increase is not observed for other interfacial regions or the whole protein. This clearly suggests that, in the E104D mutant, the large water cluster is responsible for the subunit interface flexibility and overall thermolability, and it ultimately leads to the deficiency of this enzyme.ConclusionsOur study reveals that a large cluster of water buried in protein interfaces is fragile and high-maintenance, closely related to the structure, function and evolution of the whole protein.

Dataset Information

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

Publications

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

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