Binding and internalization of an ICAM-1 peptide by the surface receptors of T cells

The objective of this work was to evaluate the binding characteristics of a cyclic peptide, cycle (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-Val9 -Thr10-Gly11-Cys12-OH (clBR), to Molt-3 T cells. This clBR peptide is deriv ed from sequence numbers 11-20 of intercellular adhesion molecule-1 (ICAM-1 ). Binding studies were performed using a fluorescence-labeled peptide (FIT C-clBR) in which the fluorescence marker fluorescein 5-isothiocyanate (FITC ) was conjugated to the N-terminal of the clBR peptide. The binding affinit y of the FITC-clBR peptide to Molt-3 T cells was evaluated using a FACScan flow cytometer. The binding specificity of the FITC-clBR peptide was also c onfirmed by inhibition of binding using unlabeled peptide (clBR). The resul ts show that FITC-clBR binds to two populations of T cells with different a ffinities; population 1 has high cell numbers (75%) but low affinity, and p opulation 2 has high binding affinity but low cell numbers (25%). Binding t o both populations was saturable and could be inhibited by the unlabeled pe ptide (clBR), suggesting a receptor-mediated binding process. In addition t o binding, receptor-mediated internalization was also observed for populati on 2; this was confirmed by confocal microscopy and temperature-dependence studies at 37 degrees C and 4 degrees C. The binding and internalization of this peptide may be carried out by surface receptors on Molt-3 T cells suc h as LFA-1. In the future, the binding and internalization of clBR peptide can be utilized as a method of targeted drug delivery to leukocytes for the treatment of leukocyte-related diseases.