DETECTION OF A GLYCOSYLATION-DEPENDENT LIGAND FOR THE T-LYMPHOCYTE CELL-ADHESION MOLECULE CD2 USING A NOVEL MULTIMERIC RECOMBINANT CD2-BINDING ASSAY

The CD2 molecule plays an important role in T cell adhesion by interac ting with the ligands CD58 (LFA-3) and CD59. In order to detect additi onal ligands for CD2, potentially of low binding affinity, we have pre pared a highly fluorescent, multimeric form of rCD2 whose binding to c ells can be quantified by flow cytometry. Initial studies demonstrated that binding of multimeric rCD2 to cells was CD2-specific, concentrat ion and time dependent, and saturable. The negative charge on cells wa s also found to play a critical role in the efficiency of multimeric r CD2 binding. Analysis of binding of multimeric rCD2 to 17 CD58+ cell t ypes revealed that only 8 of the cells exhibited binding. Failure of m ultimeric rCD2 to interact with the other cells could not be explained by differences in CD58 expression, suggesting that, in terms of CD2 b inding, there are qualitative differences in CD58 on different cell ty pes. Binding of multimeric rCD2 to six of the seven reactive cells was virtually totally inhibited by CD58 mAb pretreatment, whereas binding to the erythroleukemic line K562 was only partially blocked, suggesti ng the existence of another CD2 ligand. Subsequent studies demonstrate d that the putative new ligand is not CD59, and that it interacts with a different region of the CD2 molecule than CD58, probably a site loc ated between the T11(1) and T11(2) epitopes. The binding affinity of C D2 for the new ligand is 10-fold lower than for CD58 and, based on stu dies with truncated rCD2, the binding site for the new ligand is locat ed within the amino-terminal 105 amino acids of the CD2 polypeptide. U nlike CD58, the new ligand is tunicamycin sensitive suggesting that it contains a N-linked carbohydrate structure that is essential for func tional activity.