Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors

Shigekazu Tabata, Kimiko Kuroki, Jing Wang, Mizuho Kajikawa, Ikuo Shiratori, Daisuke Kohda, Hisashi Arase, Katsumi Maenaka

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRα) and activating (PILRβ) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance (SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILRα can bind to CD99 with low affinity (Kd ∼ 2.2 μM), but activating PILRβ binds with ∼40 times lower affinity (Kd ∼ 85 μM). In addition to our previous mutagenesis study (Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. (2008) J. Immunol. 180, 1686-1693), the SPR analysis showed that PILRα can bind to each Ala mutant of the two CD99 O-glycosylated sites (Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILRα binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRβ can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRα and PILRβ. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRα-CD99 interaction is enthalpically driven with a large entropy loss (-TΔS =- 8.9 kcal·mol-1), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.

Original languageEnglish
Pages (from-to)8893-8901
Number of pages9
JournalJournal of Biological Chemistry
Volume283
Issue number14
DOIs
Publication statusPublished - Apr 4 2008

Fingerprint

Surface Plasmon Resonance
Surface plasmon resonance
Mutagenesis
Thermodynamics
Leukocyte Rolling
Ligands
Selectins
Kinetics
T-cells
Flexible structures
Entropy
Site-Directed Mutagenesis
Infiltration
Sugars
Binding Sites
T-Lymphocytes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors. / Tabata, Shigekazu; Kuroki, Kimiko; Wang, Jing; Kajikawa, Mizuho; Shiratori, Ikuo; Kohda, Daisuke; Arase, Hisashi; Maenaka, Katsumi.

In: Journal of Biological Chemistry, Vol. 283, No. 14, 04.04.2008, p. 8893-8901.

Research output: Contribution to journalArticle

Tabata, S, Kuroki, K, Wang, J, Kajikawa, M, Shiratori, I, Kohda, D, Arase, H & Maenaka, K 2008, 'Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors', Journal of Biological Chemistry, vol. 283, no. 14, pp. 8893-8901. https://doi.org/10.1074/jbc.M709793200
Tabata, Shigekazu ; Kuroki, Kimiko ; Wang, Jing ; Kajikawa, Mizuho ; Shiratori, Ikuo ; Kohda, Daisuke ; Arase, Hisashi ; Maenaka, Katsumi. / Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 14. pp. 8893-8901.
@article{a282e33e0ae74d918279f9138c272327,
title = "Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors",
abstract = "Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRα) and activating (PILRβ) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance (SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILRα can bind to CD99 with low affinity (Kd ∼ 2.2 μM), but activating PILRβ binds with ∼40 times lower affinity (Kd ∼ 85 μM). In addition to our previous mutagenesis study (Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. (2008) J. Immunol. 180, 1686-1693), the SPR analysis showed that PILRα can bind to each Ala mutant of the two CD99 O-glycosylated sites (Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILRα binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRβ can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRα and PILRβ. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRα-CD99 interaction is enthalpically driven with a large entropy loss (-TΔS =- 8.9 kcal·mol-1), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.",
author = "Shigekazu Tabata and Kimiko Kuroki and Jing Wang and Mizuho Kajikawa and Ikuo Shiratori and Daisuke Kohda and Hisashi Arase and Katsumi Maenaka",
year = "2008",
month = "4",
day = "4",
doi = "10.1074/jbc.M709793200",
language = "English",
volume = "283",
pages = "8893--8901",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "14",

}

TY - JOUR

T1 - Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors

AU - Tabata, Shigekazu

AU - Kuroki, Kimiko

AU - Wang, Jing

AU - Kajikawa, Mizuho

AU - Shiratori, Ikuo

AU - Kohda, Daisuke

AU - Arase, Hisashi

AU - Maenaka, Katsumi

PY - 2008/4/4

Y1 - 2008/4/4

N2 - Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRα) and activating (PILRβ) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance (SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILRα can bind to CD99 with low affinity (Kd ∼ 2.2 μM), but activating PILRβ binds with ∼40 times lower affinity (Kd ∼ 85 μM). In addition to our previous mutagenesis study (Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. (2008) J. Immunol. 180, 1686-1693), the SPR analysis showed that PILRα can bind to each Ala mutant of the two CD99 O-glycosylated sites (Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILRα binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRβ can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRα and PILRβ. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRα-CD99 interaction is enthalpically driven with a large entropy loss (-TΔS =- 8.9 kcal·mol-1), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.

AB - Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRα) and activating (PILRβ) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance (SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILRα can bind to CD99 with low affinity (Kd ∼ 2.2 μM), but activating PILRβ binds with ∼40 times lower affinity (Kd ∼ 85 μM). In addition to our previous mutagenesis study (Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. (2008) J. Immunol. 180, 1686-1693), the SPR analysis showed that PILRα can bind to each Ala mutant of the two CD99 O-glycosylated sites (Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILRα binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRβ can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRα and PILRβ. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRα-CD99 interaction is enthalpically driven with a large entropy loss (-TΔS =- 8.9 kcal·mol-1), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.

UR - http://www.scopus.com/inward/record.url?scp=44049092612&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=44049092612&partnerID=8YFLogxK

U2 - 10.1074/jbc.M709793200

DO - 10.1074/jbc.M709793200

M3 - Article

C2 - 18234675

AN - SCOPUS:44049092612

VL - 283

SP - 8893

EP - 8901

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 14

ER -