Abstract
OX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, whereas OX40 is expressed on activated T cells. A construct for mouse OX40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography. Radiolabeled sCD4-OX40L bound to activated mouse T cells with a high affinity (KD = 0.2-0.4 nM) and dissociated slowly (koff = 4 x 10(-5) s-1). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcoreTM biosensor, which measures macromolecular interactions in real time. The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip. sCD4-OX40L bound the OX40 with a high affinity (KD = 3.8 nM), although this was lower than that determined on the surface of activated T cells (KD = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (KD = 190 nM) and had a relatively fast dissociation rate constant (koff = 2 x 10(-2) s-1). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 because of the low monomeric affinity.
Original language | English |
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Pages (from-to) | 5275-82 |
Number of pages | 8 |
Journal | Journal of Biological Chemistry |
Volume | 272 |
Issue number | 8 |
DOIs | |
Publication status | Published - 21 Feb 1997 |
Keywords
- Animals
- Antigens, CD27
- CHO Cells
- Cricetinae
- Gene Expression
- Gene Transfer Techniques
- Kinetics
- Lymphocyte Activation
- Membrane Glycoproteins
- Mice
- Rats
- Receptors, OX40
- Receptors, Tumor Necrosis Factor
- T-Lymphocytes
- Tumor Necrosis Factors