Inefficient peptide binding by cell-surface class II MHC molecules.
The efficiency of peptide loading onto surface class II MHC molecules in intact APC was investigated, using a previously defined europium immunoassay as well as a simplified Western blot procedure. Conditions normally employed for peptide loading in T cell stimulation assays were suboptimal for peptide binding, which is enhanced at low pH, in the presence of protease inhibitors, and the absence of competing serum proteins. In contrast to some earlier reports, our results indicate that the rate of peptide loading by class II molecules is not enhanced in the environment of the plasma membrane. Peptide association rates were similar for purified and cell-surface class II molecules. As previously reported, rapid peptide binding can be achieved by reconstituting class II molecules into total cellular membranes. We report that this activity is due solely to HLA-DM (which is not present at the cell surface), since it can be specifically removed by immunodepletion with an anti-DM mAb. Thus, we find no evidence for additional cellular cofactors capable of catalyzing peptide binding to class II molecules.
Sherman
M A
MA
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Weber
D A
DA
Spotts
E A
EA
Moore
J C
JC
Jensen
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D005063:Europium; D006681:HLA-D Antigens / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding; D013601:T-Lymphocytes / Q000276:immunology
C416189:H2-M antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D005063:Europium