Detection of low-affinity adhesion ligands by linking recombinant cell adhesion molecules in uniform orientation to a fluorescently labelled dextran 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 prepared a highly polyvalent form of CD40 attached to a dextran backbone. This was accomplished by engineering a hexahistidine tag on the C-terminus of the CD40 and binding, in a uniform orientation, up to 100 molecules of hexahistidine CD40 by metal chelation to a single fluorescently tagged dextran molecule. The advantage of this 'multimeric' CD40 is that it would be expected to bind to any counterstructure with a significantly 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 bound to the physiological ligand. Using this system, we found no evidence to support the claim [Heath et al., 1993. Cell. Immunol. 152, 468.] that the A20 cells have a counterstructure for CD40, and propose that the high binding of CD40 observed in this study may have been due to an exposed hexahistidine tag on the molecule. This multimeric technology 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.
Vassiliou
G
G
Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT. gerard.vassiliou@anu.edu.au
Jakobsen
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K
Parish
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CR
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051379:Mice; D014162:Transfection
D019013:CD40 Antigens; D015815:Cell Adhesion Molecules; D003911:Dextrans; D005456:Fluorescent Dyes; D008024:Ligands; D008562:Membrane Glycoproteins; C015219:fluorescein isothiocyanate dextran; D023201:CD40 Ligand; D006639:Histidine; D016650:Fluorescein-5-isothiocyanate