title: FRET based quantification and screening technology platform for the interactions of leukocyte function-associated antigen-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1).
abstract: The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple 'in solution' steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (Kd) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.
authors.affiliation: Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: Instituto de Carboquímica, CSIC, Zaragoza, Spain; Immune Effector Cells Group, Aragón Health Research Institute, Biomedical Research Centre of Aragón, Zaragoza, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Núñez
authors.affiliation: Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Shih-Yang
authors.initials: SY
authors.lastname: Hu
authors.affiliation: Instituto de Carboquímica, CSIC, Zaragoza, Spain; Immune Effector Cells Group, Aragón Health Research Institute, Biomedical Research Centre of Aragón, Zaragoza, Spain.
authors.forename: María Pilar
authors.initials: MP
authors.lastname: Domingo
authors.affiliation: Immune Effector Cells Group, Aragón Health Research Institute, Biomedical Research Centre of Aragón, Zaragoza, Spain; Department of Biochemistry and Molecular and Cell Biology, Facultad de Ciencias, University of Zaragoza, Zaragoza, Spain; Aragón I+D Foundation, Government of Aragon, Zaragoza, Spain; Nanoscience Institute of Aragón, Aragón I+D Foundation, University of Zaragoza, Zaragoza, Spain.
authors.forename: Julian
authors.initials: J
authors.lastname: Pardo
authors.affiliation: Biophotonics & Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Artashes
authors.initials: A
authors.lastname: Karmenyan
authors.affiliation: Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan; Biophotonics & Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Arthur
authors.initials: A
authors.lastname: Chiou
mesh_terms: D002448:Cell Adhesion / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1