Peptide induction of surface expression of class I MHC.
This unit describes a method for comparing the relative binding of different peptides to the same MHC class I (MHC-I) molecule using live cells. Live cells expressing suboptimally loaded MHC-I proteins are incubated with medium containing diluted amounts of synthetic peptides to be tested for binding to class I. After overnight incubation with peptide, surface class I expression is monitored by flow cytometry using an allele-specific MAb. Relative binding affinity of peptide reliably correlates with the amount of surface induction of the class I molecule to which it specifically binds. The mechanistic basis of this assay is that surface MHC-I molecules become conformationally unstable shortly after peptide dissociation. However, the binding of an exogenous peptide can stabilize the surface class I molecule, prevent conformational instability, and thus increase class I surface expression in an allele-specific manner.
Hansen
Ted
T
Washington University School of Medicine, St. Louis, Missouri, USA.
Myers
Nancy
N
D000483:Alleles; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology
D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D010455:Peptides