Cr. Parish et al., DETECTION OF A GLYCOSYLATION-DEPENDENT LIGAND FOR THE T-LYMPHOCYTE CELL-ADHESION MOLECULE CD2 USING A NOVEL MULTIMERIC RECOMBINANT CD2-BINDING ASSAY, The Journal of immunology, 150(11), 1993, pp. 4833-4843
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.