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Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

Westmuckett AD, Thacker KM, Moore KL - PLoS ONE (2011)

Bottom Line: We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules.Similar results were observed on immobilized P-selectin in vitro.Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

View Article: PubMed Central - PubMed

Affiliation: Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America. Andrew-Westmuckett@omrf.org

ABSTRACT

Background: We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.

Methodology/principal findings: Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

Conclusions/significance: These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

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Related in: MedlinePlus

Binding of fluid-phase P-selectin and Psgl-1 expression.Binding of P-selectin/IgM to peripheral blood leukocytes from (A) WT→B6 mice or (B) Tpst DKO→B6 mice. Shaded histograms represent binding of CD45/IgM. Binding of the anti-Psgl-1 mAb 2PH1 to peripheral blood leukocytes from (C) WT→B6 mice or (D) Tpst DKO→B6 mice. Shaded histograms represent binding of isotype control mAb. Panels A & C are same samples analyzed on the same day and are representative of 3 WT→B6 mice. Panels B & D are also same samples analyzed on the same day and are representative of 7 Tpst DKO→B6 mice. All analyses were gated on the neutrophil and monocyte population based on forward and orthogonal light scattering properties and on donor origin (CD45.2+).
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pone-0020406-g004: Binding of fluid-phase P-selectin and Psgl-1 expression.Binding of P-selectin/IgM to peripheral blood leukocytes from (A) WT→B6 mice or (B) Tpst DKO→B6 mice. Shaded histograms represent binding of CD45/IgM. Binding of the anti-Psgl-1 mAb 2PH1 to peripheral blood leukocytes from (C) WT→B6 mice or (D) Tpst DKO→B6 mice. Shaded histograms represent binding of isotype control mAb. Panels A & C are same samples analyzed on the same day and are representative of 3 WT→B6 mice. Panels B & D are also same samples analyzed on the same day and are representative of 7 Tpst DKO→B6 mice. All analyses were gated on the neutrophil and monocyte population based on forward and orthogonal light scattering properties and on donor origin (CD45.2+).

Mentions: P-selectin binding to Psgl-1 on neutrophils in peripheral blood was determined using flow cytometry. Neutrophils were gated based on their forward and orthogonal light scattering properties and on donor origin (CD45.2+). We observed that the mean fluorescence intensity (MFI) of P-selectin binding to WT→B6 cells was 1,322±138 (n = 3) and 557±64 for Tpst DKO→B6 cells (n = 7) (Fig. 4A & B). This difference is highly significant (p = 0.016). Pre-incubation of cells with P-selectin blocking mAb RB40.34 or Psgl-1 blocking mAb 4RA10 completely blocked binding of the P-selectin/IgM to levels equivalent to that for CD45/IgM (data not shown).


Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

Westmuckett AD, Thacker KM, Moore KL - PLoS ONE (2011)

Binding of fluid-phase P-selectin and Psgl-1 expression.Binding of P-selectin/IgM to peripheral blood leukocytes from (A) WT→B6 mice or (B) Tpst DKO→B6 mice. Shaded histograms represent binding of CD45/IgM. Binding of the anti-Psgl-1 mAb 2PH1 to peripheral blood leukocytes from (C) WT→B6 mice or (D) Tpst DKO→B6 mice. Shaded histograms represent binding of isotype control mAb. Panels A & C are same samples analyzed on the same day and are representative of 3 WT→B6 mice. Panels B & D are also same samples analyzed on the same day and are representative of 7 Tpst DKO→B6 mice. All analyses were gated on the neutrophil and monocyte population based on forward and orthogonal light scattering properties and on donor origin (CD45.2+).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102115&req=5

pone-0020406-g004: Binding of fluid-phase P-selectin and Psgl-1 expression.Binding of P-selectin/IgM to peripheral blood leukocytes from (A) WT→B6 mice or (B) Tpst DKO→B6 mice. Shaded histograms represent binding of CD45/IgM. Binding of the anti-Psgl-1 mAb 2PH1 to peripheral blood leukocytes from (C) WT→B6 mice or (D) Tpst DKO→B6 mice. Shaded histograms represent binding of isotype control mAb. Panels A & C are same samples analyzed on the same day and are representative of 3 WT→B6 mice. Panels B & D are also same samples analyzed on the same day and are representative of 7 Tpst DKO→B6 mice. All analyses were gated on the neutrophil and monocyte population based on forward and orthogonal light scattering properties and on donor origin (CD45.2+).
Mentions: P-selectin binding to Psgl-1 on neutrophils in peripheral blood was determined using flow cytometry. Neutrophils were gated based on their forward and orthogonal light scattering properties and on donor origin (CD45.2+). We observed that the mean fluorescence intensity (MFI) of P-selectin binding to WT→B6 cells was 1,322±138 (n = 3) and 557±64 for Tpst DKO→B6 cells (n = 7) (Fig. 4A & B). This difference is highly significant (p = 0.016). Pre-incubation of cells with P-selectin blocking mAb RB40.34 or Psgl-1 blocking mAb 4RA10 completely blocked binding of the P-selectin/IgM to levels equivalent to that for CD45/IgM (data not shown).

Bottom Line: We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules.Similar results were observed on immobilized P-selectin in vitro.Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

View Article: PubMed Central - PubMed

Affiliation: Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America. Andrew-Westmuckett@omrf.org

ABSTRACT

Background: We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.

Methodology/principal findings: Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

Conclusions/significance: These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

Show MeSH
Related in: MedlinePlus