title: Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets.
abstract: Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3-36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.
authors.affiliation: Department of Microbiology and Carter Immunology Center, University of Virginia, Charlottesville 22908, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Lippolis
authors.forename: Forest M
authors.initials: FM
authors.lastname: White
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.forename: Chance J
authors.initials: CJ
authors.lastname: Luckey
authors.forename: Timothy N J
authors.initials: TN
authors.lastname: Bullock
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Victor H
authors.initials: VH
authors.lastname: Engelhard
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017951:Antigen Presentation; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D002461:Cell Line, Transformed; D010450:Endopeptidases / Q000378:metabolism; D006684:HLA-DR Antigens / Q000032:analysis; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011499:Protein Processing, Post-Translational / Q000276:immunology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D010450:Endopeptidases