P-selectin glycoprotein ligand-1 and E-selectin ligand-1 are differentially modified by fucosyltransferases Fuc-TIV and Fuc-TVII in mouse neutrophils.
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 alpha1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid alpha1, 3-fucosyltransferases Fuc-TVII or Fuc-TIV to this alpha1, 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-TIV-deficient 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.
Huang
M C
MC
Institute of Cell Biology, ZMBE, University of Münster and Max-Planck-Institute of Physiological and Clinical Research, D-48149 Münster, Germany.
Zöllner
O
O
Moll
T
T
Maly
P
P
Thall
A D
AD
Lowe
J B
JB
Vestweber
D
D
D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009504:Neutrophils / Q000201:enzymology*; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019087:Selectins / Q000378:metabolism; D012795:Sialoglycoproteins; D014162:Transfection
D007074:Immunoglobulin G; D008024:Ligands; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D020033:Protein Isoforms; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D019087:Selectins; D012795:Sialoglycoproteins; C086711:cysteine-rich fibroblast growth factor receptor; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase