Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity
Mavaddat N, Mason DW, Atkinson PD, Evans EJ, Gilbert RJ, Stuart DI, Fennelly JA, Barclay AN, Davis SJ, Brown MH. (2000), J Biol Chem. 275, 28100-9
Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form “head to head” dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.
Key figure: Affinity of sSLAM self-association
(A) sSLAM was injected at the indicated concentrations through flow cells with immobilized sSLAMb (1273 RU) or as a negative control, 2B4CD4b (1285 RU), at 37°C. (B) The responses at equilibrium (A) in the flow cells in which sSLAMb (circles), and/or the contol protein (squares), were immobilized, and the difference between the responses (giving specific binding, C), are each plotted against the sSLAM concentration. Removal of the oligohistidine tag with carboxypeptidase A had no effect on the affinity measurements. The linear fit used to determine P (equation 6 of Experimental Procedures) is shown in (B).