Affinity and kinetics of the interaction between soluble trimeric OX40 ligand, a member of the tumor necrosis factor superfamily, and its receptor OX40 on activated T cells

被引:47
作者
AlShamkhani, A [1 ]
Mallett, S [1 ]
Brown, MH [1 ]
James, W [1 ]
Barclay, AN [1 ]
机构
[1] UNIV OXFORD,SIR WILLIAM DUNN SCH PATHOL,MRC,CELLULAR IMMUNOL UNIT,OXFORD OX1 3RE,ENGLAND
关键词
D O I
10.1074/jbc.272.8.5275
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, Radio labeled sCD4-OX40L bound to activated mouse T cells with a high affinity (K-D = 0.2-0.4 nM) and dissociated slowly (k(off) = 4 x 10(-5) s(-1)), The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcore(TM) 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 (K-D = 3.8 nM), although this was lower than that determined on the surface of activated T cells (K-D = 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 (K-D = 190 nM) and had a relatively fast dissociation rate constant (k(off) = 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.
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收藏
页码:5275 / 5282
页数:8
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