Characterisation of the low affinity interaction between rat cell adhesion molecules CD2 and CD48 by analytical ultracentrifugation
Silkowski H, Davis SJ, Barclay AN, Rowe AJ, Harding SE, Byron O. (1997), Eur Biophys J. 25, 455-62
CD2 is a cell adhesion molecule found on the plasma membrane of T-lymphocytes. Its counter-receptor in rat is the structurally related CD48. This interaction is believed to contribute to the adhesion of T-cells to other cells such as cytotoxic targets and antigen presenting cells. Cell-cell adhesion involves the formation of multiple cell adhesion molecule complexes at the cell surface and if cell-cell de-adhesion is to occur, these complexes need to be disrupted. The affinities of cell adhesion molecule interactions are suggested to be relatively weak to allow this de-adhesion of cell-cell interactions. The CD2/CD48 interaction has been studied using recombinant extracellular proteins and the affinity of the interaction of soluble recombinant rat CD2–CD48 has been determined (at 37°C) using surface plasmon resonance (and shown to be weak), with the dissociation constant Kd=60–90 µm. The values determined by surface plasmon resonance results could be affected by the immobilisation of the ligand on the chip and any self-association on the chip. We used three different analytical ultracentrifuge procedures which each allowed the interaction to be studied in free solution without the need for an immobilisation medium. Both sedimentation equilibrium (using direct analysis of the concentration distribution and also modelling of molecular weight versus concentration data) and sedimentation velocity at 5°C yielded dissociation constants in the range of 20– 110 µm, supporting the surface plasmon resonance findings showing that binding between these cell adhesion molecules is relatively weak. These studies also ruled out the presence of any significant self-association of the reactants which could lead to systematic error in the surface plasmon resonance results.
Weight-average sedimentation coefficients (●) modelled iteratively to Eqs. (7)–(11) [with ks (monomer) set as 5 ml/g; ks (dimer as 8.5 ml/g)], for values of the dissociation constant Kd in the range 8–120 μM using the software SA-Plot