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 interacting with the ligands CD58 (LFA-3) and CD59. In order to detect additional ligands for CD2, potentially of low binding affinity, we have prepared a highly fluorescent, multimeric form of rCD2 whose binding to cells can be quantified by flow cytometry. Initial studies demonstrated that binding of multimeric rCD2 to cells was CD2-specific, concentration and time dependent, and saturable. The negative charge on cells was also found to play a critical role in the efficiency of multimeric rCD2 binding. Analysis of binding of multimeric rCD2 to 17 CD58+ cell types revealed that only 8 of the cells exhibited binding. Failure of multimeric rCD2 to interact with the other cells could not be explained by differences in CD58 expression, suggesting that, in terms of CD2 binding, there are qualitative differences in CD58 on different cell types. 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, suggesting the existence of another CD2 ligand. Subsequent studies demonstrated 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 located between the T11(1) and T11(2) epitopes. The binding affinity of CD2 for the new ligand is 10-fold lower than for CD58 and, based on studies with truncated rCD2, the binding site for the new ligand is located within the amino-terminal 105 amino acids of the CD2 polypeptide. Unlike CD58, the new ligand is tunicamycin sensitive suggesting that it contains a N-linked carbohydrate structure that is essential for functional activity.
Parish
C R
CR
Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Recny
M A
MA
Knoppers
M H
MH
Waldron
J C
JC
Warren
H S
HS
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis / Q000502:physiology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D018801:CD2 Antigens; D018968:CD58 Antigens; D015815:Cell Adhesion Molecules / Q000032:analysis*; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000032:analysis / Q000502:physiology; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology; D011869:Radioligand Assay; D011971:Receptors, Immunologic / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D018968:CD58 Antigens; D015815:Cell Adhesion Molecules; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D000071228:Polyelectrolytes; D011108:Polymers; D011971:Receptors, Immunologic; D011994:Recombinant Proteins