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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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The first 19 amino acids of PSGL-1 are  sufficient for attachment to CHO-E and -P  monolayers, and the NH2-terminal anionic  polypeptide segment of PSGL-1 is necessary for  148.Fc-mediated attachment to CHO-P, but not  CHO-E monolayers. (a) Schematic representation of the PSGL-1.Fc constructs. Closed bars,  PSGL-1 segments; open bars, human Fc segments; open bar with X, an internal deletion of  amino acids 5–11 within the NH2-terminal anionic polypeptide region; shaded bar, the enterokinase cleavage site; Y, amino-terminal tyrosine;  vertical lines with open circles, the approximate  number and location of potential O-linked glycosylation sites; vertical lines with closed circles, O-linked site at amino terminal threonine 16; vertical lines with shaded rectangles, potential N-linked glycosylation sites. (Drawing not to scale.) (b) Microspheres coated with the 19.ek.Fc mutant (open bars), containing  the first 19 amino acids of PSGL-1, attached to CHO-P and -E monolayers at a rate similar to 148.Fc microspheres (dark bars). Microspheres coated with the ΔY.Fc mutant, which consists of the first 148 amino acids of PSGL-1 with an internal deletion of the residues in  positions 5–11, attached to CHO-E monolayers at a rate similar to 148.Fc microspheres but did not attach to CHO-P monolayers (crosshatched bars). (Shear stress = 2 dynes/cm2; n = 2). (c) An mAb to PSGL-1, KPL1, which requires amino acids 5–11 to recognize PSGL-1  (Table I), did not affect attachment of 148.Fc microspheres to CHO-E monolayers but eliminated attachment to CHO-P monolayers.  148.Fc microspheres treated with mAb KPL1 attached to CHO-E monolayers at a rate similar to that observed for 148.Fc microspheres  treated with control mAb KPL2 but did not attach to CHO-P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2). (d) mAb  KPL1 eliminated attachment of 19.ek.Fc microspheres to both CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2).
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Figure 6: The first 19 amino acids of PSGL-1 are sufficient for attachment to CHO-E and -P monolayers, and the NH2-terminal anionic polypeptide segment of PSGL-1 is necessary for 148.Fc-mediated attachment to CHO-P, but not CHO-E monolayers. (a) Schematic representation of the PSGL-1.Fc constructs. Closed bars, PSGL-1 segments; open bars, human Fc segments; open bar with X, an internal deletion of amino acids 5–11 within the NH2-terminal anionic polypeptide region; shaded bar, the enterokinase cleavage site; Y, amino-terminal tyrosine; vertical lines with open circles, the approximate number and location of potential O-linked glycosylation sites; vertical lines with closed circles, O-linked site at amino terminal threonine 16; vertical lines with shaded rectangles, potential N-linked glycosylation sites. (Drawing not to scale.) (b) Microspheres coated with the 19.ek.Fc mutant (open bars), containing the first 19 amino acids of PSGL-1, attached to CHO-P and -E monolayers at a rate similar to 148.Fc microspheres (dark bars). Microspheres coated with the ΔY.Fc mutant, which consists of the first 148 amino acids of PSGL-1 with an internal deletion of the residues in positions 5–11, attached to CHO-E monolayers at a rate similar to 148.Fc microspheres but did not attach to CHO-P monolayers (crosshatched bars). (Shear stress = 2 dynes/cm2; n = 2). (c) An mAb to PSGL-1, KPL1, which requires amino acids 5–11 to recognize PSGL-1 (Table I), did not affect attachment of 148.Fc microspheres to CHO-E monolayers but eliminated attachment to CHO-P monolayers. 148.Fc microspheres treated with mAb KPL1 attached to CHO-E monolayers at a rate similar to that observed for 148.Fc microspheres treated with control mAb KPL2 but did not attach to CHO-P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2). (d) mAb KPL1 eliminated attachment of 19.ek.Fc microspheres to both CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2).

Mentions: The PSGL-1 molecules used in these studies are chimeras consisting of various truncated extracellular regions of mature PSGL-1 fused to the heavy chain CH2-CH3 (Fc) region of human IgG1. Their construction, expression, and selectin-binding properties have been described previously (44). In the present studies, plasmids encoding two of these chimeras, 148.Fc and ΔY148.Fc (abbreviated to ΔY.Fc here), were transfected into COS cells as described except for the substitution of a plasmid encoding an α(1,3)fucosyltransferase-VII (Fuc-TVII) enzyme (36, 45) for the FucTIII plasmid used in earlier cotransfections (44). The resulting conditioned media were supplemented with 0.002% NaN3 and 10 μM PMSF and then clarified by centrifugation. Fc chimeras were purified by protein A–Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) chromatography per the manufacturer's instructions and quantified by an anti-Fc enzyme-linked immunosorbent assay using human IgG1 (Sigma Chemical Co.) as a standard. The 19.Fc form of PSGL-1 described earlier (44) was mutated to include an enterokinase cleavage site (19) located between the PSGL-1 and IgG Fc regions and is termed 19.ek.Fc. A plasmid encoding this construct was stably transfected into CHO cells that were engineered to also express Fuc-TVII and β(1,6)-N-acetylglucosaminyltransferase (“core2” [9]) activities (17, 25). 19.ek.Fc was purified from conditioned media and quantified as above. Similar to COS produced 19.Fc, 19.ek.Fc is tyrosine sulfated and glycosylated with SLex-type containing O-glycans and, in E- and P-selectin binding experiments (44), is functionally indistinguishable (data not shown). The nomenclature for microspheres coated with various extracellular regions of PSGL-1 is as follows: the 148.Fc construct, 148.Fc microspheres; the 19.ek.Fc construct, 19.ek.Fc microspheres; the ΔY.Fc construct, ΔY.Fc microspheres (see Fig. 6 a).


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)

The first 19 amino acids of PSGL-1 are  sufficient for attachment to CHO-E and -P  monolayers, and the NH2-terminal anionic  polypeptide segment of PSGL-1 is necessary for  148.Fc-mediated attachment to CHO-P, but not  CHO-E monolayers. (a) Schematic representation of the PSGL-1.Fc constructs. Closed bars,  PSGL-1 segments; open bars, human Fc segments; open bar with X, an internal deletion of  amino acids 5–11 within the NH2-terminal anionic polypeptide region; shaded bar, the enterokinase cleavage site; Y, amino-terminal tyrosine;  vertical lines with open circles, the approximate  number and location of potential O-linked glycosylation sites; vertical lines with closed circles, O-linked site at amino terminal threonine 16; vertical lines with shaded rectangles, potential N-linked glycosylation sites. (Drawing not to scale.) (b) Microspheres coated with the 19.ek.Fc mutant (open bars), containing  the first 19 amino acids of PSGL-1, attached to CHO-P and -E monolayers at a rate similar to 148.Fc microspheres (dark bars). Microspheres coated with the ΔY.Fc mutant, which consists of the first 148 amino acids of PSGL-1 with an internal deletion of the residues in  positions 5–11, attached to CHO-E monolayers at a rate similar to 148.Fc microspheres but did not attach to CHO-P monolayers (crosshatched bars). (Shear stress = 2 dynes/cm2; n = 2). (c) An mAb to PSGL-1, KPL1, which requires amino acids 5–11 to recognize PSGL-1  (Table I), did not affect attachment of 148.Fc microspheres to CHO-E monolayers but eliminated attachment to CHO-P monolayers.  148.Fc microspheres treated with mAb KPL1 attached to CHO-E monolayers at a rate similar to that observed for 148.Fc microspheres  treated with control mAb KPL2 but did not attach to CHO-P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2). (d) mAb  KPL1 eliminated attachment of 19.ek.Fc microspheres to both CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2).
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Figure 6: The first 19 amino acids of PSGL-1 are sufficient for attachment to CHO-E and -P monolayers, and the NH2-terminal anionic polypeptide segment of PSGL-1 is necessary for 148.Fc-mediated attachment to CHO-P, but not CHO-E monolayers. (a) Schematic representation of the PSGL-1.Fc constructs. Closed bars, PSGL-1 segments; open bars, human Fc segments; open bar with X, an internal deletion of amino acids 5–11 within the NH2-terminal anionic polypeptide region; shaded bar, the enterokinase cleavage site; Y, amino-terminal tyrosine; vertical lines with open circles, the approximate number and location of potential O-linked glycosylation sites; vertical lines with closed circles, O-linked site at amino terminal threonine 16; vertical lines with shaded rectangles, potential N-linked glycosylation sites. (Drawing not to scale.) (b) Microspheres coated with the 19.ek.Fc mutant (open bars), containing the first 19 amino acids of PSGL-1, attached to CHO-P and -E monolayers at a rate similar to 148.Fc microspheres (dark bars). Microspheres coated with the ΔY.Fc mutant, which consists of the first 148 amino acids of PSGL-1 with an internal deletion of the residues in positions 5–11, attached to CHO-E monolayers at a rate similar to 148.Fc microspheres but did not attach to CHO-P monolayers (crosshatched bars). (Shear stress = 2 dynes/cm2; n = 2). (c) An mAb to PSGL-1, KPL1, which requires amino acids 5–11 to recognize PSGL-1 (Table I), did not affect attachment of 148.Fc microspheres to CHO-E monolayers but eliminated attachment to CHO-P monolayers. 148.Fc microspheres treated with mAb KPL1 attached to CHO-E monolayers at a rate similar to that observed for 148.Fc microspheres treated with control mAb KPL2 but did not attach to CHO-P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2). (d) mAb KPL1 eliminated attachment of 19.ek.Fc microspheres to both CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; *P < 0.05; n = 2).
Mentions: The PSGL-1 molecules used in these studies are chimeras consisting of various truncated extracellular regions of mature PSGL-1 fused to the heavy chain CH2-CH3 (Fc) region of human IgG1. Their construction, expression, and selectin-binding properties have been described previously (44). In the present studies, plasmids encoding two of these chimeras, 148.Fc and ΔY148.Fc (abbreviated to ΔY.Fc here), were transfected into COS cells as described except for the substitution of a plasmid encoding an α(1,3)fucosyltransferase-VII (Fuc-TVII) enzyme (36, 45) for the FucTIII plasmid used in earlier cotransfections (44). The resulting conditioned media were supplemented with 0.002% NaN3 and 10 μM PMSF and then clarified by centrifugation. Fc chimeras were purified by protein A–Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) chromatography per the manufacturer's instructions and quantified by an anti-Fc enzyme-linked immunosorbent assay using human IgG1 (Sigma Chemical Co.) as a standard. The 19.Fc form of PSGL-1 described earlier (44) was mutated to include an enterokinase cleavage site (19) located between the PSGL-1 and IgG Fc regions and is termed 19.ek.Fc. A plasmid encoding this construct was stably transfected into CHO cells that were engineered to also express Fuc-TVII and β(1,6)-N-acetylglucosaminyltransferase (“core2” [9]) activities (17, 25). 19.ek.Fc was purified from conditioned media and quantified as above. Similar to COS produced 19.Fc, 19.ek.Fc is tyrosine sulfated and glycosylated with SLex-type containing O-glycans and, in E- and P-selectin binding experiments (44), is functionally indistinguishable (data not shown). The nomenclature for microspheres coated with various extracellular regions of PSGL-1 is as follows: the 148.Fc construct, 148.Fc microspheres; the 19.ek.Fc construct, 19.ek.Fc microspheres; the ΔY.Fc construct, ΔY.Fc microspheres (see Fig. 6 a).

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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Related in: MedlinePlus