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.
Highlights
The Leukocyte Function-associated Antigen-1 (LFA-1, known as CD11a/CD18, or aLb2), a member of the integrin superfamily of cell surface adhesion molecules, is a heterodimeric type I transmembrane glycoprotein consisting of one aL-subunit (CD11a, 180 kD) and one b2-subunit (CD18, 95 kD) which are non-covalently associated with each-other [1,2,3]
In our fluorescence resonance energy transfer (FRET) assay, the conjugated fluorophore-protein pair of Alexa Fluor 488-Leukocyte Function-Associated Antigen-1 (LFA-1) served as the donor, while Alexa Fluor 555-D1-D2-Fc as the acceptor
We have demonstrated a steady-state ‘insolution’ FRET based screening assay using the same donor and acceptor pair to identify and assess short peptides in terms of their inhibition efficiency to the LFA-1/intercellular adhesion molecule-1 (ICAM-1) interaction
Summary
The Leukocyte Function-associated Antigen-1 (LFA-1, known as CD11a/CD18, or aLb2), a member of the integrin superfamily of cell surface adhesion molecules, is a heterodimeric type I transmembrane glycoprotein consisting of one aL-subunit (CD11a, 180 kD) and one b2-subunit (CD18, 95 kD) which are non-covalently associated with each-other [1,2,3]. These two subunits form an extracellular domain, which is further subdivided into 13-subdomains, and two short cytoplasmic tails [4]. Apart from LFA-1, ICAM-1 serves as a receptor for another integrin Mac-1 [15], the human rhinovirus [16], coxsackie A21 virus [17], and malaria parasite Plasmodium falciparum [18]
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