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

Immune Network
Mi Jin LeeSun Park

Abstract

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. 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. We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to CD4(+)CD25(+) T cells similarly to the glycosyla...Continue Reading

References

Nov 16, 2005·Nature Immunology·Chen ZhuVijay K Kuchroo
May 20, 2006·The Journal of Immunology : Official Journal of the American Association of Immunologists·Sylvia Frisancho-KissDeLisa Fairweather
Aug 30, 2006·Advances in Immunology·Vijay K KuchrooRosemarie H DeKruyff
Sep 20, 2006·The Journal of Immunology : Official Journal of the American Association of Immunologists·Tsunekazu OikawaMiyuki Azuma
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Feb 20, 2008·Clinical Immunology : the Official Journal of the Clinical Immunology Society·Masako SekiMitsuomi Hirashima
Apr 1, 2008·Methods in Molecular Biology·Katalin F Medzihradszky

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Citations

Jul 6, 2016·Frontiers in Immunology·Ranferi Ocaña-GuzmanIsabel Sada-Ovalle
May 4, 2017·American Journal of Reproductive Immunology : AJRI·Stella LiongMartha Lappas
Apr 3, 2018·Journal of Immunology Research·Ranferi Ocaña-GuzmanIsabel Sada-Ovalle

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Datasets Mentioned

BETA
BAE28574.1

Methods Mentioned

BETA
glycosylation
PCR
affinity column chromatography
flow cytometry

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