L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin–Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin–Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin–Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin–Ig fusion protein was not observed. In agreement with the biochemical data, the anti–Lselectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin–Ig but not on P-selectin–Ig. No such inhibitory effect was seen with the anti–mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin–binding carbohydrate moieties are attached to different protein scaffolds in different species.
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 Eselectin, while the other only binds to P-selectin. We have analyzed the glycoprotein ligands for E-and Pselectin 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-selectinIgG; 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.
P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires ␣1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid ␣1,3-fucosyltransferases Fuc-TVII or Fuc-TIV to this ␣1,3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E-or P-selectin to PSGL-1 expressed by Fuc-TIVdeficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E-or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro ؊ 5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro ؊ 5 cells.The endothelial selectins, P-and E-selectin, are inducible adhesion molecules that initiate the tethering of granulocytes, monocytes, and subsets of lymphocytes to endothelium in inflamed tissues (1). Two major glycoprotein ligands for E-and P-selectins have been identified on mouse neutrophils (2). One of these, a 150-kDa molecule termed E-selectin ligand-1 (ESL-1) 1 (3), is recognized by E-selectin but not by P-selectin. By contrast, dimeric (230 kDa) and monomeric (130 kDa) forms of the mouse homologue of human P-selectin glycoprotein ligand-1 (PSGL-1) are recognized by both P-selectin and E-
The E-selectin ligand-1 (ESL-1) has recently been identified as the major ligand on mouse neutrophils using a recombinant antibody-like form of E-selectin as affinity probe. The remarkable selectivity with which ESL-1 can be affinity-isolated is unexplained. Since ESL-1 is endogenously expressed in Chinese hamster ovary (CHO) cells in a non-E-selectin binding form, which can become activated upon transfection of a fucosyltransferase (FucT), we analyzed various CHO cell clones, each overexpressing one of seven different fucosyltransferases, by affinity isolation experiments with E-selectin-IgG. Two of the cell lines were the regulatory CHO mutants LEC11 and LEC12, each overexpressing a different hamster FucT, while the five other clones were stably transfected with human FucTIII to -VII. A large panel of glycoproteins was affinity-isolated with E-selectin-IgG from LEC11 cells and FucTIII transfectants, demonstrating that many different glycoproteins can acquire ligand activity upon ␣(1,3)-fucosylation. In contrast, ESL-1 was almost exclusively isolated as the dominant glycoprotein ligand from LEC12 cells as well as from FucTIV and FucTVII transfectants and less selectively from FucTV and FucTVI transfectants. The selective generation of ligand activity correlated with the selective generation of the HECA452-reactive carbohydrate epitope, which is known to bind to E-selectin. These data suggest that, dependent on the type of fucosyltransferase, ESL-1 is a strongly preferred target molecule for the generation of E-selectin-binding carbohydrate modifications.
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