DETECTION OF LOW-AFFINITY ADHESION LIGANDS BY LINKING RECOMBINANT CELL-ADHESION MOLECULES IN UNIFORM ORIENTATION TO A FLUORESCENTLY LABELEDDEXTRAN MOLECULE BY MEANS OF HEXAHISTIDINE TAGGING - THE CASE OF MULTIMERIC CD40

Cell-cell interactions involve highly polyvalent associations between receptors on adjacent cells. In order to mimic this process, we have p repared a highly polyvalent form of CD40 attached to a dextran backbon e. This was accomplished by engineering a hexahistidine tag on the C-t erminus of the CD40 and binding, in a uniform orientation, up to 100 m olecules of hexahistidine CD40 by metal chelation to a single fluoresc ently tagged dextran molecule. The advantage of this 'multimeric' CD40 is that it would be expected to bind to any counterstructure with a s ignificantly higher avidity compared to monomeric CD40. The multimeric CD40 bound with high affinity to stably transfected mouse fibroblasts expressing CD40L. The multimeric ligand also bound to the activated T cell clone, D10, but did not bind to resting cells, showing that it b ound to the physiological ligand. Using this system, we found no evide nce to support the claim [Heath et al., 1993. Cell. Immunol. 152, 468. ] that the A20 cells have a counterstructure for CD40, and propose tha t the high binding of CD40 observed in this study may have been due to an exposed hexahistidine tag on the molecule. This multimeric technol ogy has considerable potential for detecting low-affinity interactions between cell adhesion receptors and ligands. The uniform orientation of the molecules on the dextran is an advantage over previous systems and permits the preparation of heterogeneous, multimeric ligands which more closely mimic the conditions at the cell surface. (C) 1998 Publi shed by Elsevier Science B.V. All rights reserved.