Antigenic peptide binding to MHC class II molecules at increased peptide concentrations.
Affinity-purified major histocompatibility complex (MHC) class II molecules are known to bind antigenic peptides in vitro. The percentage of MHC class II molecules occupied with such peptides is usually very low and varies significantly depending upon the sequence and size of a given antigenic peptide. The present study describes a method by which complete saturation of affinity-purified MHC class II with antigenic peptide can be achieved by simply incubating purified MHC class II molecules at neutral pH in the presence of several 100-fold molar excess of antigenic peptide. Complexes of human HLA-DR2 and a peptide analog from human myelin basic protein MBP (83-102)Y83 were selected for this study. The on-rate kinetic results showed saturation of MHC class II occupancy at 300-500-fold molar excess peptide concentrations. The specificity of the MBP (83-102)Y83 peptide binding to HLA-DR2 at higher peptide concentration was demonstrated by incubating an equivalent amount of another epitope from myelin basic protein [MBP (1-14) peptide] as well as by competitive binding assays. The quantitation of bound peptide was carried out using biotinylated-MBP (83-102)Y83 peptide which showed 100-125% occupancy of HLA-DR2 with a recovery of 100%. The presence of a single peptide entity in purified complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid extracted supernatant and by mass spectrometry analysis. Two-dimensional gel electrophoresis (IEF/SDS) of purified HLA-DR2 and DR2.MBP (83-102)Y83 complexes showed the absence of various endogenous polypeptides in 100% loaded complexes. These results demonstrate that higher peptide concentrations can be useful in generating MHC class II-peptide complexes of defined composition. Such complexes of MHC class II occupied with a single peptide may have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of MHC-peptide-TCR interactions.
Nag
B
B
Anergen, Inc., Redwood City, CA 94063.
Mukku
P V
PV
Arimilli
S
S
Phan
D
D
Deshpande
S V
SV
Winkelhake
J L
JL
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D015802:HLA-DR2 Antigen / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology
D000911:Antibodies, Monoclonal; D015802:HLA-DR2 Antigen; D004676:Myelin Basic Protein