Helena Yusuf-Makagiansar

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

AbstractPurpose. 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°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°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.

Synergistic inhibitory activity of α- and β-LFA-1 peptides on LFA-1/ ICAM-1 interaction

Abstract Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the α-subunit and the I-like domain of the β-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.

Sequence Recognition of α-LFA-1-derived Peptides by ICAM-1 Cell Receptors: Inhibitors of T-cell Adhesion

Blocking the T‐cell adhesion signal from intercellular adhesion molecule‐1/leukocyte function‐associated antigen‐1 interactions (Signal‐2) can suppress the progression of autoimmune diseases (i.e. type‐1 diabetes, psoriasis) and prevent allograph rejection. In this study, we determined the active region(s) of cLAB.L peptide [cyclo(1,12)Pen‐ITDGEATDSGC] by synthesizing and evaluating the biologic activity of hexapeptides in inhibiting T‐cell adhesion. A new heterotypic T‐cell adhesion assay was also developed to provide a model for the T‐cell adhesion process during lung inflammation. Two hexapeptides, ITDGEA and DGEATD, were found to be more active than the other linear hexapeptides. The cyclic derivative of ITDGEA [i.e. cyclo(1,6)ITDGEA] has similar activity than the parent linear peptide and has lower activity than cLAB.L peptide. Computational‐binding experiments were carried out to explain the possible mechanism of binding of these peptides to intercellular adhesion molecule‐1. Both ITDGEA and DGEATD bind the same site on intercellular adhesion molecule‐1 and they interact with the Gln34 and Gln73 residues on D1 of intercellular adhesion molecule‐1. In the future, more potent derivatives of cyclo(1,6)ITDGEA will be designed by utilizing structural and binding studies of the peptide to intercellular adhesion molecule‐1. The heterotypic T‐cell adhesion to Calu‐3 will also be used as another assay to evaluate the selectivity of the designed peptides.

N-cadherin involvement in the heterotypic adherence of malignant T-cells to epithelia.

N-cadherin, a cell adhesion molecule normally found in neural cell tissue, has been found recently to be expressed on the surface of malignant T-cells. The function of N-cadherin on these cells remains unclear. Heterotypic assays between Molt-3 T lymphoblastic leukemia cells and Caco-2 epithelial monolayers were examined under different conditions to assess the functional role of N-cadherin. The results indicate that adherence of Molt-3 cells to Caco-2 monolayers was reduced significantly following pretreatment of Molt-3 cells with 100 μM of an N-cadherin-derived antagonist decapeptide. In contrast, pretreatment of Molt-3 cells with an anti-N-cadherin antibody raised against the first 20 amino acids of N-cadherin sequence led to a surprisingly marked enhancement of Molt-3 cell adherence to Caco-2 monolayers. In addition, the presence of anti-N-cadherin antibody neutralized the inhibitory effect of anti-ICAM-1 on Molt-3 adhesion to Caco-2 monolayers. This novel finding demonstrates that external stimulus through the N-cadherin amino terminus can modulate adhesion of malignant T-cells to epithelia and may promote their ability to invade or metastasize to inflammatory sites.