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Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.

Goetz DJ, Greif DM, Ding H, Camphausen RT, Howes S, Comess KM, Snapp KR, Kansas GS, Luscinskas FW - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. dgoetz@cc.memphis.edu

ABSTRACT
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.

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Effect of neuraminidase treatment  of 19.ek.Fc microsphere adhesive interactions  with CHO-E and -P monolayers. Microspheres were coated with 50 μg/ml 19.ek.Fc,  treated with neuraminidase, and perfused  over CHO-E (open bars) or CHO-P (filled  bars) monolayers. (a) The rate of attachment  to CHO-E monolayers was significantly diminished, if not eliminated, by treatment with  neuraminidase. (b) In contrast, the rate of attachment to CHO-P monolayers was unaffected by neuraminidase treatment. (c) Accumulation of 19.ek.Fc microspheres on CHO-P  monolayers was significantly diminished by  treatment with neuraminidase. (Shear stress =  2 dynes/cm2; *P < 0.05; n = 3).
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Figure 7: Effect of neuraminidase treatment of 19.ek.Fc microsphere adhesive interactions with CHO-E and -P monolayers. Microspheres were coated with 50 μg/ml 19.ek.Fc, treated with neuraminidase, and perfused over CHO-E (open bars) or CHO-P (filled bars) monolayers. (a) The rate of attachment to CHO-E monolayers was significantly diminished, if not eliminated, by treatment with neuraminidase. (b) In contrast, the rate of attachment to CHO-P monolayers was unaffected by neuraminidase treatment. (c) Accumulation of 19.ek.Fc microspheres on CHO-P monolayers was significantly diminished by treatment with neuraminidase. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 3).

Mentions: To further investigate the role of glycosylation, we prepared 19.ek.Fc microspheres with 50 μg/ml 19.ek.Fc rather than 15 μg/ml and used the same neuraminidase treatment protocol. Treatment of 50 μg/ml 19.ek.Fc microspheres with neuraminidase significantly diminished, if not eliminated, attachment of 19.ek.Fc microspheres to CHO-E monolayers (Fig. 7 a). In contrast, 19.ek.Fc microspheres treated with neuraminidase attached to CHO-P monolayers at a rate similar to untreated 19.ek.Fc microspheres (Fig. 7 b). This result suggested that neuraminidase digestion of the 50 μg/ml 19.ek.Fc microspheres was incomplete, but this treatment was sufficient to abolish attachment to CHO-E but not CHO-P monolayers. Hence, a higher level of sialylation, presumably the SLex at Thr-16, is required for the 19.ek.Fc microspheres to attach to CHO-E relative to CHO-P monolayers.


Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.

Goetz DJ, Greif DM, Ding H, Camphausen RT, Howes S, Comess KM, Snapp KR, Kansas GS, Luscinskas FW - J. Cell Biol. (1997)

Effect of neuraminidase treatment  of 19.ek.Fc microsphere adhesive interactions  with CHO-E and -P monolayers. Microspheres were coated with 50 μg/ml 19.ek.Fc,  treated with neuraminidase, and perfused  over CHO-E (open bars) or CHO-P (filled  bars) monolayers. (a) The rate of attachment  to CHO-E monolayers was significantly diminished, if not eliminated, by treatment with  neuraminidase. (b) In contrast, the rate of attachment to CHO-P monolayers was unaffected by neuraminidase treatment. (c) Accumulation of 19.ek.Fc microspheres on CHO-P  monolayers was significantly diminished by  treatment with neuraminidase. (Shear stress =  2 dynes/cm2; *P < 0.05; n = 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139768&req=5

Figure 7: Effect of neuraminidase treatment of 19.ek.Fc microsphere adhesive interactions with CHO-E and -P monolayers. Microspheres were coated with 50 μg/ml 19.ek.Fc, treated with neuraminidase, and perfused over CHO-E (open bars) or CHO-P (filled bars) monolayers. (a) The rate of attachment to CHO-E monolayers was significantly diminished, if not eliminated, by treatment with neuraminidase. (b) In contrast, the rate of attachment to CHO-P monolayers was unaffected by neuraminidase treatment. (c) Accumulation of 19.ek.Fc microspheres on CHO-P monolayers was significantly diminished by treatment with neuraminidase. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 3).
Mentions: To further investigate the role of glycosylation, we prepared 19.ek.Fc microspheres with 50 μg/ml 19.ek.Fc rather than 15 μg/ml and used the same neuraminidase treatment protocol. Treatment of 50 μg/ml 19.ek.Fc microspheres with neuraminidase significantly diminished, if not eliminated, attachment of 19.ek.Fc microspheres to CHO-E monolayers (Fig. 7 a). In contrast, 19.ek.Fc microspheres treated with neuraminidase attached to CHO-P monolayers at a rate similar to untreated 19.ek.Fc microspheres (Fig. 7 b). This result suggested that neuraminidase digestion of the 50 μg/ml 19.ek.Fc microspheres was incomplete, but this treatment was sufficient to abolish attachment to CHO-E but not CHO-P monolayers. Hence, a higher level of sialylation, presumably the SLex at Thr-16, is required for the 19.ek.Fc microspheres to attach to CHO-E relative to CHO-P monolayers.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. dgoetz@cc.memphis.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus