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J. Biol. Chem., Vol. 279, Issue 1, 299-310, January 2, 2004

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Direct Measurement of Antigen Binding Properties of CD1 Proteins Using Fluorescent Lipid Probes^*

Jin S. Im, Karl O. A. Yu, Petr A. Illarionov, Kenneth P. LeClair¶, James R. Storey¶, Malcolm W. Kennedy||**, Gurdyal S. Besra, and Steven A. Porcelli

From the Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, the School of Biosciences, the University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom, ^¶Antigenics Inc., Lexington, Massachusetts 02421, and ^||Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, Graham Kerr Building, University of Glasgow, Glasgow G128QQ, Scotland, United Kingdom

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which ₂-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.

Received for publication, August 8, 2003 , and in revised form, October 6, 2003.

^* This work was supported in part by National Institutes of Health Grants 45889 and 48933 and a grant from the Irene Diamond Foundation (to S. A. P.). 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.

^** Supported by The Wellcome Trust.

A Lister Jenner Research Fellow and recipient of Medical Research Council Grants G9901077 and G0000895 and The Wellcome Trust Grants 060750 and 072021.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3226; Fax: 718-430-8711; E-mail: porcelli{at}aecom.yu.edu.

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