High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR.
Immunogenic peptides have been shown to bind detergent-solubilized class II (Ia) molecules from mice. In this investigation, we report that highly purified HLA-DR (DR) molecules in detergent solution are capable of binding a synthetic peptide (HAp) derived from the influenza hemagglutinin sequence. Although the presentation of this peptide has been demonstrated only to DR1-restricted Th cells, the association rate constants for the formation of HAp-DR1, -DR5, and -DR8 complexes were essentially identical (ka = 1.1 x 10(2) to 1.6 x 10(2) M-1 s-1). By contrast, the value of the rate constants for the dissociation of preformed HAp-DR1, -DR5, and -DR8 complexes varied nearly threefold (kd = 1.6 x 10(6) to 4.4 x 10(-6) s-1). The value of the equilibrium dissociation constants (KD) derived from these rate constants were 13 nM, 24 nM, and 28 nM, for HAp-DR1, -DR5, and -DR8 complexes, respectively. Scatchard analysis demonstrated that the KD obtained from the rate constants for the HAp-DR1 reaction was in excellent agreement with that obtained under equilibrium conditions. SDS-PAGE confirmed that the HAp-DR complexes were remarkably stable, as HAp remained associated with the DR alpha beta heterodimer after treatment of the complexes with SDS and beta-mercaptoethanol. Steady-state binding studies demonstrated that 18% of all DR1 molecules had bound HAp at equilibrium, whereas only 3.8% of all DR8 molecules had bound HAp under identical conditions. The slight differences in the KD for HAp-DR complexes suggest that differences in the affinity of a peptide for DR alleles alone may not always explain the process of MHC restriction.
Roche
P A
PA
Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
Cresswell
P
P
D000483:Alleles; D006684:HLA-DR Antigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006389:Hemagglutinins, Viral / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000276:immunology*; D007700:Kinetics; D008970:Molecular Weight; D009842:Oligopeptides / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding
D006684:HLA-DR Antigens; D006389:Hemagglutinins, Viral; D009842:Oligopeptides