Objective. To identify critical residues involved in the binding of a selective peptide to DRB1*0401.Methods. The binding of peptides to native or site-directed mutant DR molecules was evaluated using enzyme-linked immunosorbent assay and flow cytometry.Results. Amino acid substitutions at DR and peptide residues, which were predicted to contribute to interactions within the DR p4 pocket, had the greatest effects on the specificity of binding. Conclusion. Differences in the peptide-binding repertoires of DR molecules may contribute to associations with autoimmune diseases.Immune responses are determined, in part, by the assortment of HLA class I1 genes inherited by an individual (1). Recognition of peptide-class I1 molecular complexes by CD4-positive T cells initiates the cascade of events that result in an immune response and, occasionally, an autoimmune response (2). Of all the HLA class I1 molecules, those encoded by the DRBl genes are the most polymorphic (3). The polymorphic residues of the DRpl chains are located in 3 distinct hypervariable regions in the primary sequence (4). As molecular modeling studies have predicted (5,6), and crystallographic analyses of the HLA-DR1 molecule have shown (7,8), these polymorphic residues are predominantly located on one-half of the molecule, composed of both the floor (beta-pleated sheet structure) and one side (alpha-helical structure) of the peptide-binding groove. While some of the

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The intracellular trafficking, proteolysis, and dissociation of invariant chain (li) associated with nascent class II molecules was examined in B-lymphoblastoid cells. Metabolic labeling and Percoll gradient centrifugation was used to assess the kinetics of delivery and processing of class II-li complexes within the endocytic pathway. Catabolism of class II-li complexes rapidly followed their delivery from post-Golgi compartments to dense lysosome-like compartments distinct from early and late endosomes. Direct peptide binding assays revealed that class II molecules associated with even small N-terminal fragments of li failed to bind peptide. Cysteine protease inhibitors alone blocked li proteolysis/dissociation and accumulation of class II-li biosynthetic intermediates within lysosome-containing compartments. Active-site labeling of cysteine proteases in B cells was used to identify cysteine proteases capable of mediating li proteolysis within endosomal compartments. Our results indicate rapid, possibly direct, transport of nascent class II-li complexes from the Golgi/trans-Golgi network to dense lysosomal compartments wherein cysteine protease(s), likely including cathepsin B, mediate complete removal of li. Inhibition of cysteine protease activity results in the accumulation of incompletely processed class II-li complexes, which lack peptide binding ability, within lysosomal compartments.

We have synthesized a series of retro-inverso D-peptide analogues and a peptoid analog that mimic potent seven residue L-peptide ligands for DR(a. Pl*0401). The L-peptide ligands compete against binding of a 13 residue biotinylated ligand, HA307-319 (ICso 60nM), with competing peptide ICsos ranging from 30-200nM.The highest affinity heptamer retro-invcrso D-peptide tested gave ICso l0µM. No binding of the peptoid analog was detected.