Binding and internalization of an LFA-1-derived cyclic peptide by ICAM receptors on activated lymphocyte: a potential ligand for drug targeting to ICAM-1-expressing cells.
OBJECTIVE
The interaction of cell-adhesion molecules LFA-1/ICAM-1 is critical for many inflammatory and immune responses. Blockades of this interaction using antibodies or peptide analogs are being developed as therapeutic approaches for inflammatory and autoimmune diseases. The aim of this study is to examine the binding and internalization mechanisms of LFA-1 peptide [cLAB.L or cyclo-(1,12)-PenITDGEATDSGC] mediated by ICAM receptors on the surface of lymphocytes.


METHODS
The binding and internalization of cLAB.L were evaluated using fluorescence-labeled cLAB.L on activated Molt-3 cells, measured by flow cytometry. Confocal fluorescence microscopy was also used to image the distribution of peptide binding and internalization.


RESULTS
The binding of FITC-cLAB.L exhibited bimodal cell distribution and was enhanced by Ca2+ and Mg2+. Marked differences in peptide binding were found between 37 and 4 degrees C, as well as between activated and non-activated cells. Unlabeled peptide, low temperature, and the absence of cell activation suppress the peptide binding. The presence of peptide in the cytoplasm was detected in 37 but not 4 degrees C binding. Peptide cLAB.L inhibited the binding of monoclonal antibodies to domain D1 of ICAM-1 and domain D1 of ICAM-3.


CONCLUSIONS
Peptide cLAB.L can bind to the D1-domain of ICAM-1 and, to a lesser extent, to ICAM-3 on activated T-cells. Peptide binding indicates responses to the multiple and dynamic states of activated receptor ICAMs, this peptide may also be internalized by ICAM receptors on T-cells. This work suggests that cLAB.L has a therapeutic potential to target drugs to ICAM-1 expressing cells including autoreactive lymphocytes and inflamed tissues.
Yusuf-Makagiansar
H
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Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 60047, USA.
Siahaan
T J
TJ
D002412:Cations / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D008213:Lymphocyte Activation / Q000502:physiology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D013696:Temperature
D002412:Cations; D015815:Cell Adhesion Molecules; D016169:Lymphocyte Function-Associated Antigen-1; D010455:Peptides; D018799:Intercellular Adhesion Molecule-1