Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.
Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.
Goetz
D J
DJ
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. dgoetz@cc.memphis.edu
Greif
D M
DM
Ding
H
H
Camphausen
R T
RT
Howes
S
S
Comess
K M
KM
Snapp
K R
KR
Kansas
G S
GS
Luscinskas
F W
FW
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D019040:E-Selectin / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D008863:Microspheres; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D000080506:Sialyl Lewis X Antigen; D013912:Threonine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D019040:E-Selectin; D007141:Immunoglobulin Fc Fragments; D008024:Ligands; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; D000080506:Sialyl Lewis X Antigen; D014409:Tumor Necrosis Factor-alpha; D013912:Threonine; D009439:Neuraminidase; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase