An N-linked glycan modulates the interaction between the CD1d heavy chain and β2-microglobulin

Abstract

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with β2- microglobulin (β2m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to β2m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-β2m heterodimers. None of the four were indispensable for assembly or the ability to bind α-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present α-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(α1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-β2m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with β2m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from β2m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from β2m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and β2m, which may explain these unusual properties. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.