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O-Glycans Direct Selectin Ligands to Lipid Rafts on Leukocytes

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O-Glycans Direct Selectin Ligands to Lipid Rafts on Leukocytes O-glycans direct selectin ligands to lipid rafts on leukocytes Bojing Shaoa, Tadayuki Yagoa, Hendra Setiadia, Ying Wanga,b, Padmaja Mehta-D’souzaa, Jianxin Fua, Paul R. Crockerc, William Rodgersb, Lijun Xiaa,b, and Rodger P. McEvera,b,1 aCardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; bDepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and cDivision of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom Edited by Philip W. Majerus, Washington University Medical School, St. Louis, MO, and approved May 11, 2015 (received for review April 21, 2015) Palmitoylated cysteines typically target transmembrane proteins deceleration (slow rolling), arrest, intraluminal crawling, and trans- to domains enriched in cholesterol and sphingolipids (lipid rafts). endothelial migration (7). Selectins mediate rolling, whereas β2 P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are integrins mediate slow rolling, arrest, and crawling. Selectins O-glycosylated proteins on leukocytes that associate with lipid rafts. are lectins that form rapidly reversible, force-regulated bonds During inflammation, they transduce signals by engaging selectins with glycosylated ligands under flow (8). Leukocytes express as leukocytes roll in venules, and they move to the raft-enriched L-selectin, activated platelets express P-selectin, and activated uropods of polarized cells upon chemokine stimulation. It is not endothelial cells express P- and E-selectin. The dominant leuko- known how these glycoproteins associate with lipid rafts or whether cyte ligand for P- and L-selectin is P-selectin glycoprotein ligand-1 this association is required for signaling or for translocation to (PSGL-1). Major leukocyte ligands for E-selectin include PSGL-1, uropods. Here, we found that loss of core 1-derived O-glycans in CD44, and CD43, although other ligands contribute to adhesion murine C1galt1−/− neutrophils blocked raft targeting of PSGL-1, (9). PSGL-1 and CD43 are mucins with multiple O-glycans at- CD43, and CD44, but not of other glycosylated proteins, as mea- tached to serines and threonines. Although not a mucin, CD44 is sured by resistance to solubilization in nonionic detergent and by modified with both N- and O-glycans (10, 11). The selectins bind, in part, to the sialyl Lewis x (sLex) determinant (NeuAcα2– copatching with a raft-resident sphingolipid on intact cells. Neur- β – α – β aminidase removal of sialic acids from wild-type neutrophils also 3Gal 1 4[Fuc 1 3]GlcNAc 1-R), which caps some N-glycans CELL BIOLOGY − − blocked raft targeting. C1galt1 / neutrophils or neuraminidase- and mucin-type O-glycans (8, 12). CD44 uses N-glycans to in- teract with E-selectin (13, 14), whereas PSGL-1 uses mucin-type, treated neutrophils failed to activate tyrosine kinases when plated core 1-derived O-glycans to interact with all three selectins (14–16). on immobilized anti–PSGL-1 or anti-CD44 F(ab′)2. Furthermore, − − The enzyme core 1 β1–3-galactosyltransferase forms the core 1 C1galt1 / neutrophils incubated with anti–PSGL-1 F(ab′) did 2 backbone (Galβ1–3GalNAcα1-Ser/Thr) to which more distal de- not generate microparticles. In marked contrast, PSGL-1, CD43, terminants such as sLex are added (17). Neutrophils from mice and CD44 moved normally to the uropods of chemokine-stimu- − − lacking core 1 β1–3-galactosyltransferase in endothelial and he- / − − lated C1galt1 neutrophils. These data define a role for core 1- matopoietic cells (EHC C1galt1 / ) have markedly impaired derived O-glycans and terminal sialic acids in targeting glycopro- rolling on P- or E-selectin (14). tein ligands for selectins to lipid rafts of leukocytes. Preassociation PSGL-1 (18, 19), CD44 (20), and CD43 (21) associate with of these glycoproteins with rafts is required for signaling but not lipid rafts on leukocytes, but how they do so is unclear. In knockin for movement to uropods. mice, PSGL-1 lacking the cytoplasmic domain still associates with leukocyte rafts (19). In transfected nonhematopoietic cells, cell adhesion | cell signaling | inflammation | uropod | neutrophil Significance ipid rafts are ordered membrane domains that assemble Lcholesterol, sphingolipids, and selected proteins (1). They Leukocytes partition certain proteins into cholesterol- and were first defined by resistance to solubilization in cold nonionic sphingolipid-rich membrane regions (lipid rafts) that function detergents, which maintains raft proteins in the lighter fractions as signaling platforms. Inflammatory stimuli cause leukocytes of density gradients (2). On intact cells, lateral crosslinking with to elongate to form lamellipodia and uropods at opposite ends antibodies or other probes copatches lipid and protein constit- that facilitate migration. Many raft-associated proteins move uents of rafts (3). High-resolution imaging confirms that rafts are to uropods. Proteins are typically thought to use their trans- small, dispersed structures that can be oligomerized (1). Im- membrane and cytoplasmic domains to associate with rafts. portantly, rafts serve as signaling platforms, notably on immune Here, we found that some leukocyte adhesion proteins used cells (4). carbohydrate modification (glycosylation) of their extracellular How proteins partition to rafts is incompletely understood (5). domains to associate with lipid rafts. These proteins re- Hydrophobic residues in some transmembrane domains may quired preassociation with rafts to transduce signals but, un- interact with sphingolipids and/or cholesterol. Cysteines modi- expectedly, not to move to uropods. These data define a mech- fied with saturated fatty acids, usually palmitic acid, direct cy- anism for localizing proteins to critical membrane regions of tosolic proteins such as Src family kinases (SFKs) to raft inner leukocytes. leaflets. Palmitoylated cysteines in transmembrane and cytoplas- mic domains of some membrane proteins also interact with rafts. Author contributions: B.S., T.Y., P.R.C., W.R., L.X., and R.P.M. designed research; B.S., T.Y., In polarized epithelial cells, apical transport vesicles are enriched H.S., Y.W., and P.M.-D. performed research; J.F., P.R.C., and L.X. contributed new re- in cholesterol and sphingolipids (1). N- and O-glycans on some agents/analytic tools; B.S., T.Y., H.S., Y.W., P.M.-D., J.F., P.R.C., W.R., L.X., and R.P.M. apical proteins act as sorting determinants, probably through analyzed data; and B.S. and R.P.M. wrote the paper. multiple mechanisms. Glycans may enhance association of some Conflict of interest statement: R.P.M. has equity interest in Selexys Pharmaceuticals apically destined proteins with rafts (6). Whether glycans direct Corporation. proteins to rafts of hematopoietic cells is unknown. This article is a PNAS Direct Submission. At sites of infection or injury, circulating leukocytes adhere 1To whom correspondence should be addressed. Email: [email protected]. to activated endothelial cells and platelets and to adherent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. leukocytes. The adhesion cascade includes tethering, rolling, 1073/pnas.1507712112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1507712112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 detergent resistance of CD44 is reversed by mutating cysteines in PSGL-1 or CD44 from activating SFKs and generating micro- the transmembrane and cytoplasmic domains and an ezrin/rad- particles. However, O-glycans were not required to redistribute ixin/moesin (ERM)-binding site in the cytoplasmic domain (22). PSGL-1, CD43, or CD44 to the uropods of polarized leukocytes. Whether similar mechanisms operate in primary leukocytes is unknown. Determining how these proteins target to rafts is rele- Results vant because of their important signaling functions in leuko- PSGL-1 Does Not Require Its Transmembrane Domain to Associate cytes. Selectin binding to PSGL-1 or CD44 on neutrophils triggers with Lipid Rafts. Deleting the cytoplasmic domain of PSGL-1 phosphorylation of SFKs and downstream mediators that convert does not prevent its partitioning into detergent-resistant mem- β2 integrins to an extended, intermediate-affinity state, slowing branes (DRMs, lipid rafts) (19). We asked whether PSGL-1 re- rolling and contributing to arrest (23–25). Disrupting lipid rafts by quires its transmembrane domain to associate with rafts. We depleting or sequestering cholesterol blocks signaling (23). Binding generated PSGL-1 chimeras that substituted the transmembrane of P-selectin to PSGL-1 on myeloid cells causes shedding of mi- domain of PSGL-1 with the transmembrane domain of glyco- croparticles with proinflammatory and procoagulant properties phorin A or of CD45, which do not partition into rafts (18, 32, (26). The microparticles are enriched in raft-associated proteins 33) (Fig. S1A). Wild-type (WT) PSGL-1 and PSGL-1 chimeras such as PSGL-1 and tissue factor, but not in nonraft proteins such were expressed in transfected Chinese hamster ovary cells at as CD45 (18). Disrupting rafts by chelating or sequestering cho- similar densities (Fig. S1B). The cells were also transfected with lesterol blocks microparticle generation (18). However, it is not vectors that express glycosyltransferases required to construct known whether PSGL-1 or other selectin ligands must preasso- selectin ligands (34). We lysed the cells in
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