O-glycans direct selectin ligands to lipid rafts on leukocytes

Abstract

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1−/− neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1−/− neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti–PSGL-1 or anti-CD44 F(ab′)2. Furthermore, C1galt1−/− neutrophils incubated with anti–PSGL-1 F(ab′)2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1−/− neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Prominin-1/CD133: Lipid Raft Association, Detergent Resistance, and Immunodetection

“…In CD34 1 cell-derived EVs, CD133 showed the same susceptibility to Triton X-100 extraction already at 0.1% (Fig. 1B), while CD162 was partly associated with the Triton X-100resistant membrane, as previously reported [37]. Flotillins marked also resistance to Triton X-100 solubilization while moesin, an adapter protein linking the plasma membrane to the cytoskeleton, was associated with the soluble fraction irrespective of the detergent (Fig.…”

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

“…39 Previous models of TNF-or thioglycollate-induced inflammation may have overestimated the ability of PTx to block chemokine signaling in endogenous neutrophils for prolonged periods. 11,27,28,37,39,41,45,49 This could explain why some signaling pathways attributed to selectins, notably activation of PI3Kg, could instead be caused by chemokines. Although we cannot exclude some contribution of selectins to activating PI3Kg in neutrophils, our data indicate that chemokine-induced signals are dominant.…”

PSGL-1: A New Player in the Immune Checkpoint Landscape

“…Much of what is known about signaling mechanisms that can be engaged by PSGL-1 comes from studies of neutrophils. In the absence of selectin binding, PSGL-1 localizes along with integrins in lipid rafts that are distributed over the surface of circulating leukocytes [45]. Upon adhesion to the endothelium, leukocytes adopt a polarized shape in response to chemokine gradients regulated by Rho GTPases [46].…”