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

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

doi:10.1074/jbc.m308803200

Abstract

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 beta2-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.

Highlights

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses
This observation led us to modify the structure of CD1 proteins by covalently linking ␤2m to the N termini of CD1 via a flexible (G4S)3 linker to create fully assembled secreted CD1 proteins with a single chain CD1 structure
Stable transfectants expressing Single chain CD1 (scCD1) proteins were cultivated by using low protein, serum-free medium that enhanced the quality of purification by Ni-NTA-agarose chromatography

Summary

Introduction

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. Four classes of lipid antigens are known to be presented by CD1 molecules to T cells as follows: 1) mycolates (including free mycolic acids and glucose monomycolates from mycobacteria and related bacteria [6]); 2) diacylglycerols (mycobacterial lipoarabinomannans, phosphatidylinositols, and eukaryotic glycosylphosphatidylinositols [5, 11]); 3) glycosphingolipids (gangliosides, sulfatides, and ␣-glycosylceramides [12,13,14]); and 4) glycosylphosphoisoprenoids (mycobacterial mannosylated phosphoisoprenoids [4]) All of these lipid antigens contain a common motif of a polar head group covalently linked to one or more hydrophobic alkyl chains. The unique structure of mycobacterial lipid antigens presented by CD1c, which are mannosylated phosphoisoprenoids that contain a single alkyl tail with multiple methyl branches, suggests that the ligand binding region of CD1c may have slightly different properties than those of CD1b and CD1d

Methods

Results

Conclusion