PHOTOAFFINITY-LABELING OF THE T-CELL RECEPTOR ON CLONED CYTOTOXIC T-LYMPHOCYTES BY COVALENT PHOTOREACTIVE LIGAND

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LUESCHER, IF
CEROTTINI, JC
ROMERO, P
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Q5 [生物化学]; Q7 [分子生物学];
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071010 ; 081704 ;
摘要
The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2K(d) (K-d) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The K-d-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble K-d molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent K-d-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-K-d antibody and was absent on K-d-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.
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页码:5574 / 5582
页数:9
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