Rapid nonlysosomal degradation of assembled HLA class II glycoproteins incorporating a mutant DR alpha-chain.
Gamma irradiation followed by antibody and complement selection was used to isolate a human B-lymphoblastoid cell line that no longer expresses HLA-DR molecules on its cell surface. Cell surface expression in the mutant (HMy2.DRN) was restored by transfecting a wildtype DRA but not a DRB cDNA, suggesting that a structural mutation in the DRA mRNA or protein was responsible for the lack of cell surface expression. Nucleotide sequence analysis of the DRA mRNA from HMy2.DRN revealed a 75 nucleotide deletion corresponding to the start of the alpha 2 domain and involving one of two cysteines that are involved in the formation of an intrachain disulfide bond. At the biochemical level, only minute quantities of HLA-DR could be precipitated from this cell line after a 4-h continuous label with 35S-methionine. HLA-DR beta and the class II-associated invariant chain could be seen coprecipitating with the mutant DR alpha-chain, suggesting a limited accumulation of normally assembled molecules. However, by carrying out the labeling at 16 degrees C instead of 37 degrees C, equivalent amounts of HLA-DR could be precipitated from parent and mutant alike. The mutant DR alpha chain was found in association with the beta-chain, but with reduced association with the invariant chain under these conditions. Pulse chase analysis in the parent and mutant cell lines indicated that this mutant DR alpha beta I complex undergoes a process of degradation at 37 degrees C. Inhibitors of intracellular transport such as monensin were ineffective in blocking this process of degradation. This work is consistent with published reports implicating the involvement of a pre-Golgi or an early Golgi compartment in the proteolysis of aberrantly folded or assembled multisubunit proteins.
Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
B
B
Koppelman
P
P
Cresswell
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002451:Cell Compartmentation; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D006684:HLA-DR Antigens / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D009154:Mutation
D006684:HLA-DR Antigens; D004247:DNA; D008985:Monensin