Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48

van der Merwe PA, Brown MH, Davis SJ, Barclay AN. (1993), EMBO J. 12, 4945-54

Abstract

CD2 is a plasma membrane glycoprotein present on T lymphocytes that functions 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 cells and de-adhesion requires disruption of these complexes. To gain an insight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore instrument, which enables the monitoring of protein binding in real time. A soluble chimeric protein, comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4), bound to immobilized soluble CD2 (sCD2) with a KD of 90 microM. The affinity was also determined in the reverse orientation and sCD2 was shown to bind immobilized sCD48-CD4 with a comparable KD of 60 microM. sCD48-CD4 bound to immobilized deglycosylated sCD2 with a KD of 125 microM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The low affinity was the result of an extremely rapid off-rate constant (K(off) > or = 6 s-1), whereas the on-rate constant was unremarkable (K(on) > or = 10(5) M-1s-1). The kinetic analysis revealed that small amounts of multimeric aggregates of sCD48-CD4 formed in concentrated preparations. Our experience suggests that even low concentrations (< 2%) of these aggregates may be a cause of artifactually high affinity values when analysing low-affinity protein interactions. In conclusion, this study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.

Key figure: Analysis of multimeric aggregates in sCD48-CD4 preparation

(A) Either sCD48-CD4 (10 mg, ●) or sCD2 (15 mg, ▲) were loaded onto a 200 ml Sephadex G-75 column in a 1 ml sample and 2 ml fractions were collected at 6 ml/h. OX34 IgG contaminating the sCD2 sample (which was purified on an OX34 affinity column) eluted near the void (IgG). OX34 Fab eluted between sCD48-CD4 and sCD2 (Fab). (B) The binding to immobilized sCD2 of: BSA at 0.6 mg/ml for 15 s; unfractionated sCD48-CD4 (pre-GF) at 0.6 mg/ml for 15 s; the monomeric sCD48-CD4 peak (#37) at 0.6 mg/ml for 15 s; the multimeric peak (#31) at 0.025 mg/ml for 60 s. (C) Effect of crosslinking sCD48-CD4 on binding to immobilized sCD2. Injection for 120 s of monomeric sCD48-CD4 at 50 μg/ml, OX68 at 300 μg/ml, or both sCD48-CD4 plus OX68 at 50 and 300 μg/ml, respectively. In (A) and (B) bound sCD48-CD4 was eluted with 0.1 M HCl (arrows).