Novel Glycosylation of HLA-DRα Disrupts Antigen Presentation Without Altering Endosomal Localization

Author:

Guerra Carolyn B.1,Busch Robert2,Doebele Robert C.12,Liu Wendy2,Sawada Tetsuji3,Kwok William W.4,Chang Ming-der Y.3,Mellins Elizabeth D.2

Affiliation:

1. *School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

2. †Department of Pediatrics, Stanford University Medical Center, Stanford, CA 94305;

3. ‡Department of Medicine, North Shore University Hospital-New York University School of Medicine, Manhasset, NY 11030; and

4. §Virginia Mason Research Center, Seattle, WA 98101

Abstract

AbstractThe 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 Ala96α, 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.

Publisher

The American Association of Immunologists

Subject

Immunology,Immunology and Allergy

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