The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated.
The HECA452 carbohydrate epitope, also termed cutaneous lymphocyte antigen, is known to bind to E-selectin and defines a human T cell subset preferentially found in inflamed skin. Activated T cells can express a functional form of the P-selectin glycoprotein ligand-1 (PSGL-1), the major ligand known for P-selectin. Here we show that PSGL-1 can exist in two forms, of which only one carries the HECA452 epitope and binds to E-selectin, while the other only binds to P-selectin. We have analyzed the glycoprotein ligands for E- and P-selectin on the mouse CD8+ T cell clone 4G3 at 4, 8, and 12 days after antigen-specific activation. Only at day 4 did the cells bind to E-selectin, whereas cells at all three activation stages bound to P-selectin. Expression of the HECA452 epitope correlated with E-selectin binding. In affinity isolation experiments, PSGL-1 was isolated as the major ligand by E-selectin-IgG and by P-selectin-IgG; however, PSGL-1 only bound to E-selectin at day 4, whereas it bound to P-selectin at all three activation stages. Immunoprecipitated PSGL-1 from cells at day 4, but not from cells at days 8 and 12, was recognized in immunoblots by monoclonal antibody HECA452. In immunoblots of total extracts of cells at day 4, HECA452 recognized a 240/140-kDa pair of protein bands as the major antigen. These bands could be completely removed by depletion of cell extracts with anti-PSGL-1 antibodies. Our data suggest that the carbohydrate requirements for binding of PSGL-1 to P-selectin differ from those necessary for binding to E-selectin. Furthermore, we conclude that the major glycoprotein carrier for the HECA452 epitope on activated 4G3 cells is PSGL-1.
Borges
E
E
Institute of Cell Biology, ZMBE, University of Münster, D-48149 Münster, Germany.
Pendl
G
G
Eytner
R
R
Steegmaier
M
M
Zöllner
O
O
Vestweber
D
D
D000818:Animals; D050260:Carbohydrate Metabolism; D019040:E-Selectin / Q000378:metabolism*; D018604:Epitope Mapping; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D009077:Mucins / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D019040:E-Selectin; D007074:Immunoglobulin G; D008024:Ligands; D008562:Membrane Glycoproteins; D009077:Mucins; D019007:P-Selectin; C084882:P-selectin ligand protein