AFFINITY AND KINETIC-ANALYSIS OF THE INTERACTION OF THE CELL-ADHESIONMOLECULES RAT CD2 AND CD48

CD2 is a plasma membrane glycoprotein present on T lymphocytes that fu nctions as a cell adhesion molecule (CAM). The CD2 counter-receptor in rodents is the structurally-related CAM CD48. Intercellular adhesion involves the formation of multiple CAM complexes between adhering cell s and de-adhesion requires disruption of these complexes. To gain an i nsight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore(TM) instrument, which enables the monitoring of prote in binding in real time. A soluble chimeric protein, comprising the ex tracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48- CD4), bound to immobilized soluble CD2 (sCD2) with a K(D) of 90 muM. T he affinity was also determined in the reverse orientation and sCD2 wa s shown to bind immobilized sCD48-CD4 with a comparable K(D) of 60 muM . sCD48-CD4 bound to immobilized deglycosylated sCD2 with a K(D) of 12 5 muM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The tow affinity was the result of an ext remely rapid off-rate constant (K(off) greater-than-or-equal-to 6 s-1) , whereas the on-rate constant was unremarkable (K(on) greater-than-or -equal-to 10(5) M-1s-1). The kinetic analysis revealed that small amou nts of multimeric aggregates of sCD48-CD4 formed in concentrated prepa rations. Our experience suggests that even low concentrations (< 2%) o f these aggregates may be a cause of artefactually high affinity value s when analysing low-affinity protein interactions. In conclusion, thi s study provides the first detailed analysis of the kinetics and affin ity of monomeric CAM interactions and suggests that binding between CA Ms may be weaker than anticipated.