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

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.

Figure 1. Sequence alignment of the N-terminal P-selectin binding sites of human and mouse P-selectin glycoprotein ligand-1.

Figure 2. P-selectin dependent rolling in vivo.

Surgical exteriorization of the cremaster muscle was used to induce P-selectin-dependent leukocyte rolling in post-capillary venules. (A) The rolling flux fraction in 49 venules from 7 WT→B6 mice and 39 venules from 7 Tpst DKO→B6 mice was determined. (B) Mean rolling velocities were determined from an average of 8.5 leukocytes/venule in the same venules as the rolling flux fractions. All values are reported as mean ± S.E.M.

Figure 3. P-selectin dependent leukocyte rolling in vitro.

Bone marrow leukocytes from wild type (n = 3) and Tpst DKO→B6 (n = 3) mice were isolated and their rolling on immobilized mouse P-selectin/IgM (site density  = 100 sites/µm²) was observed at 1 dyn/cm². (A) For each animal, the number of rolling cells in 4 fields of view were averaged. (B) Rolling velocities of 10 leukocytes were determined in the same 4 fields of view as the number of rolling cells. Values are reported as mean ± S.E.M.

Figure 4. 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⁺).