Antigen-processing organelles from DRB1*1101 and DRB1*1104 B cell lines display a differential degradation activity.
We have developed an in vitro assay for tetanus toxin (tt) C fragment (C-fr) degradation. Purified endosomes (abbreviated endosomes 1101 or 1104) and lysosomes (abbreviated lysosomes 1101 or 1104) from the DRB1*1101 (Gly 86) and DRB1*1104 (Val 86) B cell lines were used to degrade 125I-labeled C-fr in vitro. Using three distinct methods of analysis, we show that the capacity of endosomes and lysosomes to degrade the tt C-fr or tt synthetic Y-P30 peptide differed. Using sodium dodecylsulfate-polyacrylamide gel electrophoresis, 125I-labeled C-fr degradation patterns observed either with endosomes 1101/1104 or lysosomes 1101/1104 are distinct both in terms of the number of fragments and the kinetics of generation of the fragments. These results were confirmed by high-performance liquid chromatography analysis, where we observed that the elution profiles of the 125I-labeled Y-P30 peptide digested by endosomes 1101/1104 were different compared to those obtained with lysosomes 1101/1104. Furthermore, the kinetics of degradation of 125I-labeled Y-P30 were faster with lysosomes 1104 than with lysosomes 1101. This difference in activity of the 1101 and 1104 organelles was also found in a functional assay where we showed that the activation capacity of the P30 peptide was diminished when digested by lysosome 1104, regardless of the antigen-presenting cell (APC) used, whereas endosomes 1101 or lysosomes 1101 modified P30 peptide in a form that discriminated between presentation by 1101 or 1104 APC. Taken together, these results suggest that the differential processing and presentation displayed by the DRB1*1101 and DRB1*1104 APC is due partly to a different enzymatic content and partly to the dimorphism at position DR beta 86.
Institute of Biochemistry, University of Lausanne, Switzerland.
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Barbey
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Watts
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Corradin
D000595:Amino Acid Sequence; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D000941:Antigens / Q000378:metabolism*; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D011992:Endosomes / Q000276:immunology; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006801:Humans; D008247:Lysosomes / Q000276:immunology; D008969:Molecular Sequence Data; D015388:Organelles / Q000276:immunology*; D010446:Peptide Fragments / Q000378:metabolism; D013744:Tetanus Toxin / Q000378:metabolism
D000941:Antigens; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; C091828:P30 tetanus toxin peptide; D010446:Peptide Fragments; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C