Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48.
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.
van der Merwe
P A
PA
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Brown
M H
MH
Davis
S J
SJ
Barclay
A N
AN
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000378:metabolism*; D018801:CD2 Antigens; D015704:CD4 Antigens / Q000378:metabolism; D000071178:CD48 Antigen; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D015704:CD4 Antigens; D000071178:CD48 Antigen; C000604067:Cd48 protein, rat; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic