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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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A PSGL-1.Fc chimera molecule can be coupled to protein A microspheres. 10-μm microspheres were precoated with  protein A. These microspheres were then incubated with various  concentrations of the 148.Fc chimera. The amount of 148.Fc chimera bound to the microspheres was detected with an mAb to  PSGL-1 (a–e), PSL-275, and an appropriate FITC-labeled secondary antibody. Isotype-matched control mAb to ICAM-1,  Hu5/3, did not recognize the 148.Fc chimera (f   ).
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Figure 1: A PSGL-1.Fc chimera molecule can be coupled to protein A microspheres. 10-μm microspheres were precoated with protein A. These microspheres were then incubated with various concentrations of the 148.Fc chimera. The amount of 148.Fc chimera bound to the microspheres was detected with an mAb to PSGL-1 (a–e), PSL-275, and an appropriate FITC-labeled secondary antibody. Isotype-matched control mAb to ICAM-1, Hu5/3, did not recognize the 148.Fc chimera (f   ).

Mentions: In initial studies, 148.Fc, a truncated PSGL-1 construct consisting of the first 148 amino acids of PSGL-1 linked to the Fc region of human IgG1 (44), was coupled to 10-μmdiam polystyrene microspheres precoated with protein A to generate 148.Fc microspheres. The 148.Fc version of PSGL-1 was used, as it was demonstrated previously to exhibit high affinity binding to both E- and P-selectin under static conditions when produced in the presence of an SLex-generating fucosyltransferase (44). We chose to use 10-μm-diam microspheres since such microspheres have a density (1.05 g/ml) and diameter similar to that of leukocytes. The presence of the 148.Fc chimera on the surface of the 148.Fc microsphere was detected with an mAb to PSGL-1, PSL-275 (Fig. 1, a–e). The amount of 148.Fc chimera bound to the microspheres was concentration dependent (Fig. 1, a–e). An isotype-matched control mAb, Hu5/3, did not recognize microspheres coated with 100 μg/ml 148.Fc, the highest coating concentration used (Fig. 1 f). From this data we conclude that the 148.Fc chimera was bound to the protein A–coated microspheres. Preliminary studies indicated that microspheres prepared with 50 μg/ml of the 148.Fc construct gave consistent results in the flow adhesion assays. Hence, a coating concentration of 50 μg/ml was used in the remainder of the studies unless otherwise noted.


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)

A PSGL-1.Fc chimera molecule can be coupled to protein A microspheres. 10-μm microspheres were precoated with  protein A. These microspheres were then incubated with various  concentrations of the 148.Fc chimera. The amount of 148.Fc chimera bound to the microspheres was detected with an mAb to  PSGL-1 (a–e), PSL-275, and an appropriate FITC-labeled secondary antibody. Isotype-matched control mAb to ICAM-1,  Hu5/3, did not recognize the 148.Fc chimera (f   ).
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Related In: Results  -  Collection

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

Figure 1: A PSGL-1.Fc chimera molecule can be coupled to protein A microspheres. 10-μm microspheres were precoated with protein A. These microspheres were then incubated with various concentrations of the 148.Fc chimera. The amount of 148.Fc chimera bound to the microspheres was detected with an mAb to PSGL-1 (a–e), PSL-275, and an appropriate FITC-labeled secondary antibody. Isotype-matched control mAb to ICAM-1, Hu5/3, did not recognize the 148.Fc chimera (f   ).
Mentions: In initial studies, 148.Fc, a truncated PSGL-1 construct consisting of the first 148 amino acids of PSGL-1 linked to the Fc region of human IgG1 (44), was coupled to 10-μmdiam polystyrene microspheres precoated with protein A to generate 148.Fc microspheres. The 148.Fc version of PSGL-1 was used, as it was demonstrated previously to exhibit high affinity binding to both E- and P-selectin under static conditions when produced in the presence of an SLex-generating fucosyltransferase (44). We chose to use 10-μm-diam microspheres since such microspheres have a density (1.05 g/ml) and diameter similar to that of leukocytes. The presence of the 148.Fc chimera on the surface of the 148.Fc microsphere was detected with an mAb to PSGL-1, PSL-275 (Fig. 1, a–e). The amount of 148.Fc chimera bound to the microspheres was concentration dependent (Fig. 1, a–e). An isotype-matched control mAb, Hu5/3, did not recognize microspheres coated with 100 μg/ml 148.Fc, the highest coating concentration used (Fig. 1 f). From this data we conclude that the 148.Fc chimera was bound to the protein A–coated microspheres. Preliminary studies indicated that microspheres prepared with 50 μg/ml of the 148.Fc construct gave consistent results in the flow adhesion assays. Hence, a coating concentration of 50 μg/ml was used in the remainder of the studies unless otherwise noted.

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