Siglec-E is a negative regulator of acute pulmonary neutrophil inflammation and suppresses CD11b β2-integrin–dependent signaling

McMillan, Scott; Sharma, Ritu; McKenzie, Emma; Richards, Hannah; Zhang, Jiquan; Prescott, Alan R.; Crocker, Paul R.

doi:10.1182/blood-2012-08-449983

Cited by 105 publications

(150 citation statements)

References 45 publications

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“…Siglec-E, the siglec CD33, and PILRα are expressed on murine myeloid cells. However, all three lectins have cytoplasmic immunoreceptor tyrosine-based inhibitory motifs that negatively regulate inflammation (43,44), whereas raft association of PSGL-1, CD43, and CD44 promotes proinflammatory signaling. CD33 and PIRLα prefer sialic acid linked α2-6 to N-acetylgalactosamine (45,46), not the sialic acid linked α2-3 to galactose that caps core 1-derived O-glycans.…”

Section: Discussionmentioning

confidence: 99%

O-glycans direct selectin ligands to lipid rafts on leukocytes

Shao

Yago

Setiadi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionmentioning

confidence: 99%

O-glycans direct selectin ligands to lipid rafts on leukocytes

Shao

Yago

Setiadi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Removal of sialic acids with sialidase Neu1 can disrupt this interaction, resulting in activation of inflammatory processes. LPS stimulation induces exaggerated recruitment of neutrophils in the lung in Siglec-E knock-out mice (14,15). In mouse models of bacterial infection (16), tumor growth (17), and sepsis (18), Siglec-E plays a critical role in the pathogenesis of diseases.…”

Section: Cd33-related Siglecs Are a Family Of Proteins Widely Expressmentioning

confidence: 99%

“…Many of these studies deciphering the role of Siglec-E involved the use of Siglec-E knock-out mouse (14). No blocking/non-blocking antibody pair specific to Siglec-E is available at present.…”

Section: Cd33-related Siglecs Are a Family Of Proteins Widely Expressmentioning

confidence: 99%

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E

Siddiqui

Schwarz

Springer

et al. 2017

Journal of Biological Chemistry

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Edited by Amanda F. Fosang CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation.Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer. Two broad groups of Siglecs are identified based on their patterns of evolution. Sialoadhesin, CD22, myelin-associated glycoprotein (MAG) and Siglec-15 are highly conserved and show few changes among species (2). In contrast, CD33-related Siglecs evolve rapidly in mammals (2, 9). As there are not clear functional orthologs between primates and rodent Siglecs, these receptors are assigned an alphabetic letter in rodents and a number in primates. Siglec-E, the main CD33-related Siglec in mouse, was first discovered by a yeast two-hybrid screen using SHP-1 as a bait (10). Studies using a sheep polyclonal Siglec-E antibody specific for the ectodomain indicated that Siglec-E is expressed on neutrophils, macrophages, monocytes, dendritic cells, and a subset of Natural killer cells (11). The organ with the * This work was supported, in whole or in part, by National Institutes of Health Grants P01HL107150 and R01GM32373 (to A. V.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. □ S Th...

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“…The LGALS3BP-deficient mice showed an increased reactivity to LPS, which suggests a hyperactive myeloid system (64). In this regard, Siglec-E is expressed on myelomonocytic cells and has also been shown previously to dampen the response to LPS (65). Although murine LGALS3BP binds to Siglec-E (Fig.…”

Section: Discussionmentioning

confidence: 95%

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

Läubli

Alisson-Silva

Stanczak

et al. 2014

Journal of Biological Chemistry

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Background: Engagement of inhibitory CD33-related Siglecs on immune cells has been shown to influence interactions with cancer cells, including tumor immune evasion. Results:LGALS3BP binds with high affinity to CD33-related Siglecs and inhibits neutrophil activation. Conclusion: We identify LGALS3BP as novel, cancer-associated Siglec ligand that can influence neutrophil activation. Significance: The engagement of inhibitory CD33-related Siglecs by LGALS3BP could support immune evasion of tumor cells.

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Siglec-E is a negative regulator of acute pulmonary neutrophil inflammation and suppresses CD11b β2-integrin–dependent signaling

Abstract: Key Points First report describing in vivo function of siglec-E as a negative regulator of neutrophil recruitment in acute lung inflammation. Implications for the human functional ortholog, siglec-9, and its potential role in regulating inflammatory lung disease.

Cited by 105 publications

References 45 publications

O-glycans direct selectin ligands to lipid rafts on leukocytes

O-glycans direct selectin ligands to lipid rafts on leukocytes

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

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