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© The Rockefeller University Press,
0021-9525/1997//509 $5.00
The Journal of Cell Biology, Volume 137, Number 2,
, 1997 509-519
Article |
Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin
Small Molecule Drug Discovery Group, Genetics Institute, Cambridge, Massachusetts 02140; and Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611
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-µm-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-
–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.
1. Abbreviations used in this paper: CHO-E, and -P, CHO cell monolayers stably expressing E- or P-selectin; DPBS, Dulbecco's phosphate buffered saline; Fuc-TVII,
(1,3)fucosyltransferase-VII; HUVEC, human umbilical vein endothelial cell(s); OSGE, O-sialoglycoprotein endopeptidase; PSGL-1, P-selectin glycoprotein ligand-1; RT, room temperature; SLex, sialyl Lewis x; TNF-, tumor necrosis factor-. This research was supported by The National Cancer Institute grant F32CA71129, National Institutes of Health (NIH) training grant T32HL07627 (D.J. Goetz), and NIH grants HL36028 and HL53993 (F.W. Luscinskas) and by a grant from the American Cancer Society (G.S. Kansas). G.S. Kansas is an Established Investigator at the American Heart Association. D.M. Greif was supported by the Stanley J. Sarnoff Endowment for Cardiovascular Science, Inc.
Douglas J. Goetz's present address is The University of Memphis, Department of Biomedical Engineering, Engineering Technology Building Room 330, Memphis, TN 38152. Tel.: (901) 678-8675. Fax: (901) 678-5281. E-mail: dgoetz{at}cc.memphis.edu
Douglas J. Goetz and Daniel M. Greif contributed equally to this work.
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