Identification of core structure and critical T cell receptor contact residues in an antigenic peptide by measuring acidification rates.
A silicon-based biosensor microphysiometer measures real time cell response by monitoring an increase in extracellular acidification rate in response to ligands for specific membrane receptors. We used the microphysiometer to identify the minimal structure and critical residues of an antigenic peptide for its interaction with T cell receptor (TCR) using a synthetic peptide analog of human myelin basic protein (MBP) corresponding to residues 84-102 [MBP(83-102)Y83]. MBP(83-102)Y83 peptide analogs were allowed to interact with TCRs on a DRB5*0101-restricted Herpes virus saimiri (HVS) transformed human T cell clone (SS8T) which also contains major histocompatibility complexes (MHC) class II (DR2) molecules. Cultured SS8T cells were exposed to 11 N-terminus and 11 C-terminus truncated peptides separately in the microphysiometer chambers to determine the minimal amino acid residues required for the T cell response. In parallel, 13 analogs of the MBP(83-102)Y83 peptide with single alanine substitutions were tested in this assay to identify critical amino acid residues involved in TCR interactions. A minimal core length of MBP(91-100) peptide and residues F-91, K-93, N-94, I-95 and V-96 were essential for TCR interaction. Acidification rate measurements correlated well with enhanced levels of gamma-IFN (interferon gamma) and TNF-beta cytokine production and suggested that the increase in the extracellular acidification rate is a direct result of early T cell signaling events.
Anergen, Redwood City, CA 94063, USA.
S
S
Arimilli
S V
SV
Deshpande
B
B
Nag
D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D002999:Clone Cells; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology*; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*
D004676:Myelin Basic Protein; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; C113463:myelin basic protein 83-102