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-l (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 463 cells is PSGL-1.