title: Novel glycosylation of HLA-DRalpha disrupts antigen presentation without altering endosomal localization.
abstract: The HLA-DR hemizygous B lymphoblastoid cell line, 10.24.6, has a DRA mutation (Pro96-->Ser) that creates a novel glycosylation site at Asn94. The mutant DR molecules are primarily associated with nested fragments of invariant chain (class II-associated invariant chain peptides), and their interaction with HLA-DM is impaired. Here we further analyzed the defect in 10.24.6 cells. Expressing Ser96 mutant DRA cDNA in DRA-null cells recapitulated the 10.24.6 phenotype, indicating that the mutation causes the Ag presentation defect. A mutation to Ala96alpha, which does not introduce an extra glycan, generated a normal phenotype; the critical role of the glycan was further supported by experiments in which N-glycosylation was blocked by tunicamycin. We also evaluated whether the 10.24.6 mutation affected DR3 maturation or trafficking. Metabolic labeling and subcellular fractionation showed that assembly, endosomal transport, and invariant chain proteolysis of mutant DR3 molecules were similar to wild-type. A slight delay in export from the endoplasmic reticulum to the Golgi apparatus in 10.24.6 cells probably did not contribute significantly to the Ag presentation defect, because the abundance of DM and mutant DR in peptide-loading compartments was normal at steady state. Our results indicate that proper localization of these molecules does not depend on their interaction.
authors.affiliation: School of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
authors.forename: C B
authors.initials: CB
authors.lastname: Guerra
authors.forename: R
authors.initials: R
authors.lastname: Busch
authors.forename: R C
authors.initials: RC
authors.lastname: Doebele
authors.forename: W
authors.initials: W
authors.lastname: Liu
authors.forename: T
authors.initials: T
authors.lastname: Sawada
authors.forename: W W
authors.initials: WW
authors.lastname: Kwok
authors.forename: M D
authors.initials: MD
authors.lastname: Chang
authors.forename: E D
authors.initials: ED
authors.lastname: Mellins
mesh_terms: D017951:Antigen Presentation / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology* / Q000648:ultrastructure; D001692:Biological Transport / Q000276:immunology; D002460:Cell Line; D011992:Endosomes / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006684:HLA-DR Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D009154:Mutation
chemical_list: D006684:HLA-DR Antigens