The goal of this proposal is to characterize the regulatory T-cell protein lymphocyte activation gene-3 (LAG- 3). The LAG-3 protein plays an important role in negatively regulating T-cell activation and proliferation;and in its soluble form has the power to activate antigen presenting cells. This protein is expressed in activated natural killer cells, CD4+ and CD8+ T-cells. B-cells also express LAG-3 in a T-cell dependent manner. LAG- 3 is a CD4-homolog that, like CD4, binds to class II MHC proteins. A dimeric state of LAG-3 is observed on the cell surface of activated T-cells, but very little is known about the protein after it is cleaved from the cell surface, which is known as soluble LAG-3. We propose here experiments to: a) complete ongoing efforts to obtain refolded and functional LAG-3 protein, b) determine the biophysical properties of the oligomeric state of sLAG-3 as well as that of the interaction of LAG-3 with class II MHC proteins, c) determine the interaction site of possible LAG-3 oligomers and that of LAG-3 with MHC II molecules. We will continue our efforts in producing soluble functional LAG-3 by purifying it from inclusion bodies and refolding it in vitro as well as by expressing it in Drosphila Schneider-2 cells. Characterization of LAG-3 oligomers and the LAG-3:MHC II complex will be done by gel filtration to determine the size and molecular mass under native conditions, dynamic light scattering to determine the size distribution and the translational diffusion coefficient D, as well as cross linking studies to characterize the oligomeric state of sLAG-3. Affinity constants of the binding of LAG-3 with class II MHC proteins will be examined by gel filtration and dynamic light scattering while kinetic and affinity parameters of the interaction will be studied by surface plasmon resonance. The three-dimensional structure of LAG-3 by itself and in complex with a class II MHC will be obtained by x-ray crystallography to determine the oligomeric state of LAG-3, and the interaction site between these two molecules;any potential conformational changes on LAG-3 upon MHC II binding will be examined as well. Differential chemical modifications of a class II MHC protein in the presence and absence of LAG-3 of the complex will be performed. Understanding the biochemical characteristics of LAG-3 will facilitate the identification of the mechanism by which this protein regulates T-cell activation and is capable of activating the most potent antigen presenting cells, dendritic cells.
