The interaction affinity between vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) analyzed by quantitative FRET.

Sandeep Chakraborty,Shu-Han Wu,Artashes Karmenyan,Shih-Yang Hu,Arthur Chiou,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0121399

Abstract

Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an ‘in solution’ steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (Kd). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, Kd of this interaction was determined to be 41.82 ± 2.36 nM. To further confirm our estimation, we have employed surface plasmon resonance (SPR) technique to obtain Kd = 39.60 ± 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based ‘in solution’ simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of Kd can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions.

Highlights

Protein-protein interactions play a critical role in a wide variety of cellular processes
When there is no binding between Very Late Antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), only one emission peak at 520 nm will appear when the mixture of protein conjugates were excited at 470 nm
We report the successful application of “in solution” fluorescence resonance energy transfer (FRET) based quantitative assay to obtain the equilibrium dissociation constant, Kd, of the interaction between VLA-4 and VCAM-1

Summary

Introduction

Protein-protein interactions play a critical role in a wide variety of cellular processes One such interacting protein pair, Vascular Cell Adhesion Molecule-1 (VCAM-1/CD106) and Very Late Antigen-4 (VLA-4), is essential in the interactions of leukocytes with cytokine-activated endothelial cells during transendothelial migrations [1,2,3] as well as in the formation of immunological synapse [4, 5]. Two major natural ligands of VLA-4 are the alternatively spliced connecting segment (CS-1) of fibronectin [9] and VCAM-1; the latter is a cytokine activated endothelial cell surface receptor protein [10, 11]. The I-like domain of the β-subunit forms the head domain in VLA-4 and harbors a metal-ion-dependent-adhesion-site (MIDAS), which binds the divalent cations, as mentioned before, necessary for binding to VCAM-1 [16]. It is interesting to note that binding of VCAM-1 to D4 needs VLA-4 activation (by either divalent cations or chemokines), whereas the binding to D1 does not [18]

Methods

Results

Conclusion