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J. Biol. Chem., Vol. 283, Issue 14, 8893-8901, April 4, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/14/8893    most recent M709793200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Tabata, S. Articles by Maenaka, K. PubMed PubMed Citation Articles by Tabata, S. Articles by Maenaka, K. Social Bookmarking                      What's this?

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 Kohda¹, Hisashi Arase^¶1, and Katsumi Maenaka¹²

From the Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, the Department of Immunochemistry, Research Institute for Microbial Diseases, and the ^¶World Premier International Immunology Frontier Research Center, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, and Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILR) 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 PILR can bind to CD99 with low affinity (K_d 2.2 µM), but activating PILRβ binds with 40 times lower affinity (K_d 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 PILR 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 PILR 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 PILR 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 PILR-CD99 interaction is enthalpically driven with a large entropy loss (–TS = 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.

Received for publication, November 30, 2007 , and in revised form, January 28, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Tables S1 and S2.

¹ Supported by the Ministry of Education, Science, Sports, Culture and Technology of Japan.

² To whom correspondence should be addressed. Tel.: 81-92-642-6969; Fax: 81-92-642-6764; E-mail: kmaenaka{at}bioreg.kyushu-u.ac.jp.

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