An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-microglobulin.
Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). 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 beta(2)m 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-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m 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 beta(2)m, which may explain these unusual properties.
Paduraru
Crina
C
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.
Spiridon
Laurentiu
L
Yuan
Weiming
W
Bricard
Gabriel
G
Valencia
Xavier
X
Porcelli
Steven A
SA
Illarionov
Petr A
PA
Besra
Gurdyal S
GS
Petrescu
Stefana M
SM
Petrescu
Andrei-Jose
AJ
Cresswell
Peter
P
D000595:Amino Acid Sequence; D017951:Antigen Presentation / Q000235:genetics; D018949:Antigens, CD1 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055610:Antigens, CD1d; D002236:Carbohydrate Conformation; D002999:Clone Cells; D019281:Dimerization; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D007694:Killer Cells, Natural / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011134:Polysaccharides / Q000737:chemistry* / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000378:metabolism*
D018949:Antigens, CD1; D055610:Antigens, CD1d; C526877:CD1D protein, human; D011134:Polysaccharides; D021122:Protein Subunits; D001613:beta 2-Microglobulin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase