title: 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.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
authors.forename: Bojing
authors.initials: B
authors.lastname: Shao
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Yago
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
authors.forename: Hendra
authors.initials: H
authors.lastname: Setiadi
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104;
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
authors.forename: Padmaja
authors.initials: P
authors.lastname: Mehta-D'souza
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
authors.forename: Jianxin
authors.initials: J
authors.lastname: Fu
authors.affiliation: Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Crocker
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104;
authors.forename: William
authors.initials: W
authors.lastname: Rodgers
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104;
authors.forename: Lijun
authors.initials: L
authors.lastname: Xia
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; rodger-mcever@omrf.org.
authors.forename: Rodger P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000818:Animals; D018960:Hyaluronan Receptors / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism*; D051917:Leukosialin / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: C476399:Cd44 protein, mouse; D018960:Hyaluronan Receptors; D051917:Leukosialin; D008024:Ligands; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D011134:Polysaccharides
keywords: cell adhesion; cell signaling; inflammation; neutrophil; uropod