The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2
van der Merwe PA, McPherson DC, Brown MH, Barclay AN, Cyster JG, Williams AF, Davis SJ. (1993), Eur J Immunol. 23, 1373-7
CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentrifugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 x 10(5) M-1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.
Key figure: sCD48-CD4-coated Dynabeads rosetting on CD2-transfected COS-7 cells
Rat CD2-transfected COS-7 cells were incubated with (A) sCD48-CD4- or (B) sCD4d3+4-coated Dynabeads, washed, and then counterstained with methylene blue. (C) For mAb inhibition of Dynabead rosetting, COS-7 cells expressing CD2 were pre-incubated for 1 h with OX34 TCS, OX1 TCS, or RPMI/10% fetal calf serum (No mAb) before adding sCD48-CD4-coated (hatched bars) or sCD4d3+4-coated (filled bar) Dynabeads in the same medium. For OX45 inhibition, sCD48-CD4 Dynabeads were pre-incubated for 1 h with OX45 TCS. Dynabead rosetting was quantitated as described in Sect. 2.