Peptide binding to class I molecules of the major histocompatibility complex on the surface of living target cells.
Molecules encoded by the class I major histocompatibility genes bind short (nonameric) peptides produced by intracellular proteolysis of antigens. These complexes formed intracellularly are then expressed on membranes of target cells and recognized by the antigen receptor of cytolytic T cells. No binding of externally added peptides could so far be monitored directly on the antigen presenting cells, although cytotoxicity experiments and indirect binding assays provided evidence for its existence. Here we report experiments where specific binding to class I molecules, of externally added peptides, has been monitored on living cells. N-terminal biotin-labelled Kd-restricted peptides (residues 147-155, residues 147-158, and an analogue lacking the arginine at position 156, derived from the sequence of the influenza A virus nucleoprotein) were incubated with murine H-2Kd mastocytoma cells (line P815) at 4 degrees C. The binding on surface of live, intact cells was then demonstrated fluorometrically via the interaction of a streptavidin-phycoerythrin conjugate with the biotin-labelled peptides. Thus, this binding does not involve processing, and its specificity in terms of peptide structure was established by competition with the respective unmodified peptides. The specificity of binding to class I molecules was demonstrated by blocking experiments using monoclonal antibodies specific for H-2Kd. Finally, a correlation was observed between the results of peptide binding measurements and those of cytotoxicity assays.
Kubitscheck
U
U
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Levi
R
R
Horwitz
R J
RJ
Arnon
R
R
Pecht
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D010455:Peptides