RESUMEN
Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.
Asunto(s)
Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Análisis de Matrices Tisulares/métodos , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Mucina-1 , Polisacáridos/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.
Asunto(s)
Enfermedad de Alzheimer , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Sulfato de Queratano/metabolismo , Ligandos , Ratones , Microglía/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismoRESUMEN
BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al ADN/inmunología , Lectinas/inmunología , Proteínas Supresoras de Tumor/inmunología , Bronquios/inmunología , Proteínas de Unión al Calcio/química , Proteínas de Unión al ADN/química , Eosinófilos/inmunología , Humanos , Ligandos , Mastocitos/inmunología , Líquido del Lavado Nasal/inmunología , Proteoglicanos/inmunología , Tráquea/inmunología , Proteínas Supresoras de Tumor/químicaRESUMEN
The Siglec family of cell surface receptors have emerged as attractive targets for cell-directed therapies due to their restricted expression on immune cells, endocytic properties, and ability to modulate receptor signaling. Human Siglec-8, for instance, has been identified as a therapeutic target for the treatment of eosinophil and mast cell disorders. A promising strategy to target Siglecs involves the use of liposomal nanoparticles with a multivalent display of Siglec ligands. A key challenge for this approach is the identification of a high affinity ligand for the target Siglec. Here, we report the development of a ligand of Siglec-8 and its closest murine functional orthologue Siglec-F that is capable of targeting liposomes to cells expressing Siglec-8 or -F. A glycan microarray library of synthetic 9-N-sulfonyl sialoside analogues was screened to identify potential lead compounds. The best ligand, 9-N-(2-naphthyl-sulfonyl)-Neu5Acα2-3-[6-O-sulfo]-Galß1-4GlcNAc (6'-O-sulfo NSANeu5Ac) combined the lead 2-naphthyl sulfonyl C-9 substituent with the preferred sulfated scaffold. The ligand 6'-O-sulfo NSANeu5Ac was conjugated to lipids for display on liposomes to evaluate targeted delivery to cells. Targeted liposomes showed strong in vitro binding/uptake and selectivity to cells expressing Siglec-8 or -F and, when administered to mice, exhibit in vivo targeting to Siglec-F+ eosinophils.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/metabolismo , Linfocitos B/efectos de los fármacos , Lectinas/antagonistas & inhibidores , Ácidos Siálicos/farmacología , Sulfonamidas/farmacología , Linfocitos T/efectos de los fármacos , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Células CHO , Cricetulus , Humanos , Lectinas/metabolismo , Liposomas/química , Liposomas/metabolismo , Ratones , Conformación Molecular , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Ácidos Siálicos/química , Sulfonamidas/química , Linfocitos T/metabolismoRESUMEN
Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.
Asunto(s)
Antígenos CD/química , Antígenos de Diferenciación de Linfocitos B/química , Sulfato de Queratano/química , Lectinas/química , Proteoglicanos/química , Ácidos Siálicos/química , Tráquea/química , Antígenos CD/aislamiento & purificación , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/aislamiento & purificación , Antígenos de Diferenciación de Linfocitos B/metabolismo , Apoptosis/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Sulfato de Queratano/metabolismo , Sulfato de Queratano/farmacología , Lectinas/aislamiento & purificación , Lectinas/metabolismo , Ligandos , Masculino , Persona de Mediana Edad , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , Ácidos Siálicos/metabolismo , Ácidos Siálicos/farmacología , Tráquea/metabolismoRESUMEN
Siglecs are transmembrane sialoglycan binding proteins, most of which are expressed on leukocyte subsets and have inhibitory motifs that translate cell surface ligation into immune suppression. In humans, Siglec-8 on eosinophils, mast cells and basophils and Siglec-9 on neutrophils, monocytes and some T-cells, mediate immune cell death, inhibition of immune mediator release and/or enhancement of anti-inflammatory mediator release. Endogenous sialoglycan ligands in tissues, mostly uncharacterized, engage siglecs on leukocytes to inhibit inflammation. Glycan array analyses demonstrated that Siglec-8, Siglec-9 and their mouse counterparts Siglec-F and Siglec-E (respectively) have distinct glycan binding specificities, with Siglec-8 more structurally restricted. Since siglecs are involved in lung inflammation, we studied Siglec-8 and Siglec-9 ligands in human lungs and airways. Siglec-8 ligands are in tracheal submucosal glands and cartilage but not airway epithelium or connective tissues, whereas Siglec-9 ligands are broadly distributed. Mouse airways do not have Siglec-8 ligands, whereas Siglec-9 ligands are on airways of both species. Extraction of human airways and lung followed by electrophoretic resolution and siglec blotting revealed Siglec-8 ligands in extracts of human trachea and cultured tracheal gland cells, but not parenchyma or cultured airway epithelial cells whereas Siglec-9 ligands were extracted from all airway and lung tissues and cells tested. Siglec-8 and Siglec-9 ligands in airways appear to be high molecular weight O-linked sialoglycoproteins. These data reveal differential glycan specificities of Siglec-8, Siglec-9 and their mouse counterparts Siglec-F and Siglec-E, and the tissue distributions and molecular characteristics of Siglec-8 and Siglec-9 sialoglycan ligands on human airways and lungs.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Lectinas/metabolismo , Mucosa Respiratoria/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Adulto , Antígenos CD/química , Antígenos de Diferenciación de Linfocitos B/química , Células Cultivadas , Femenino , Humanos , Lectinas/química , Ligandos , Pulmón/citología , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Tráquea/citología , Tráquea/metabolismoRESUMEN
BACKGROUND: Balanced activation and inhibition of the immune system ensures pathogen clearance while avoiding hyperinflammation. Siglecs, sialic acid-binding proteins found on subsets of immune cells, often inhibit inflammation: Siglec-8 on eosinophils and Siglec-9 on neutrophils engage sialoglycan ligands on airways to diminish ongoing inflammation. The identities of human siglec ligands and their expression during inflammation are largely unknown. OBJECTIVE: The histologic distribution, expression, and molecular characteristics of siglec ligands were explored in healthy and inflamed human upper airways and in a cellular model of airway inflammation. METHODS: Normal and chronically inflamed upper airway tissues were stained for siglec ligands. The ligands were extracted from normal and inflamed tissues and from human Calu-3 cells for quantitative analysis by means of siglec blotting and isolation by means of siglec capture. RESULTS: Siglec-8 ligands were expressed on a subpopulation of submucosal gland cells of human inferior turbinate, whereas Siglec-9 ligands were expressed more broadly (submucosal glands, epithelium, and connective tissue); both were significantly upregulated in patients with chronic rhinosinusitis. Human airway (Calu-3) cells expressed Siglec-9 ligands on mucin 5B (MUC5B) under inflammatory control through the nuclear factor κB pathway, and MUC5B carried sialoglycan ligands of Siglec-9 on human upper airway tissue. CONCLUSION: Inflammation results in upregulation of immune-inhibitory Siglec-8 and Siglec-9 sialoglycan ligands on human airways. Siglec-9 ligands are upregulated through the nuclear factor κB pathway, resulting in their enhanced expression on MUC5B. Siglec sialoglycan ligand expression in inflamed cells and tissues may contribute to the control of airway inflammation.
Asunto(s)
Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos B/genética , Células Epiteliales/inmunología , Lectinas/genética , Mucosa Respiratoria/inmunología , Rinitis/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Sinusitis/genética , Adulto , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Línea Celular , Enfermedad Crónica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica , Humanos , Lectinas/inmunología , Ligandos , Lipopolisacáridos , Masculino , Persona de Mediana Edad , Mucina 5B/antagonistas & inhibidores , Mucina 5B/genética , Mucina 5B/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Rinitis/inmunología , Rinitis/patología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Transducción de Señal , Sinusitis/inmunología , Sinusitis/patología , Factor de Necrosis Tumoral alfaRESUMEN
Legionaminic acids are analogs of sialic acid that occur in several bacteria. The most commonly occurring form is Leg5Ac7Ac, which differs from Neu5Ac only at the C7 (acetamido) and C9 (deoxy) positions. While these differences greatly reduce the susceptibility of Leg compounds to sialidases, several sialyltransferases have been identified that can use CMP-Leg5Ac7Ac as a donor (Watson et al. 2011). We report the successful modification with Leg5Ac7Ac of a glycolipid, GM1a, and two glycoproteins, interferon-α2b and α1-antitrypsin, by means of two mammalian sialyltransferases, namely porcine ST3Gal1 and human ST6Gal1. The Leg5Ac7Ac form of GD1a was not recognized by the myelin-associated glycoprotein (MAG, Siglec-4), confirming the importance of the glycerol moiety in the interaction of sialo-glycans with Siglecs.
Asunto(s)
Ácidos Siálicos/química , Sialiltransferasas/química , Animales , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Humanos , Interferón-alfa/química , Interferón-alfa/metabolismo , Glicoproteína Asociada a Mielina/química , Glicoproteína Asociada a Mielina/metabolismo , Unión Proteica , Sialiltransferasas/metabolismo , Porcinos , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismoRESUMEN
Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25±10Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH2 was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-c[Asp-His-2'-D-Nal-Arg-Trp-Lys]-NH2 and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)3 units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.
Asunto(s)
Oligopéptidos/química , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Secuencia de Aminoácidos , Células HEK293 , Humanos , Cinética , Elementos de la Serie de los Lantanoides/química , Ligandos , Hormonas Estimuladoras de los Melanocitos/química , Hormonas Estimuladoras de los Melanocitos/metabolismo , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Unión Proteica , Receptor de Melanocortina Tipo 4/metabolismoRESUMEN
We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na(2)SO(4) reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.
Asunto(s)
Citomegalovirus/enzimología , Endopeptidasas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Virales/metabolismo , Dominio Catalítico , Cromatografía de Afinidad/métodos , Endopeptidasas/aislamiento & purificación , Humanos , Serina Endopeptidasas/aislamiento & purificación , Proteínas Virales/aislamiento & purificaciónRESUMEN
O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer's disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Increasing the stoichiometry of O-GlcNAc on proteins before analysis Basic Protocol 2: Detection of proteins modified by O-GlcNAc using antibodies Basic Protocol 3: Detection of proteins modified by O-GlcNAc using the lectin sWGA Support Protocol 1: Control for O-linked glycosylation Basic Protocol 4: Detection and enrichment of proteins using WGA-agarose Support Protocol 2: Digestion of proteins with hexosaminidase Alternate Protocol: Detection of proteins modified by O-GlcNAc using galactosyltransferase Support Protocol 3: Autogalactosylation of galactosyltransferase Support Protocol 4: Assay of galactosyltransferase activity Basic Protocol 5: Characterization of labeled glycans by ß-elimination and chromatography Basic Protocol 6: Detection of O-GlcNAc in 96-well plates Basic Protocol 7: Assay for OGT activity Support Protocol 5: Desalting of O-GlcNAc transferase Basic Protocol 8: Assay for O-GlcNAcase activity.
Asunto(s)
Acetilglucosamina , Diabetes Mellitus Tipo 2 , Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicosilación , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
AIMS: Siglec-E, the mouse ortholog of human Siglec-9, is an immunosuppressive cell surface receptor. Both Siglec-E and Siglec-9 are primarily found on neutrophils, macrophages, and monocytes. When Siglec-E binds to sialoglycan ligands in its extracellular environment, it halts the immune cells' inflammatory responses. In the present study, we aimed to investigate expression, mechanisms of action and regulation of Siglec-E ligands during vascular inflammation induced by E. coli lipopolysaccharides (LPS) in mouse aorta. METHODS: The distribution, molecular size and glycoprotein class of Siglec-E ligands on mouse aorta were determined, and the protein carrier of the ligands was identified. In vivo, the expression of Siglec-E ligands was detected after LPS treatment, with or without NF-κB inhibitor administration. In vitro, cultured primary mouse aortic endothelial cells (MAECs) were used to study changes in expression of Siglec-E ligands induced by LPS with or without NF-κB inhibitors. MAECs induced by LPS were co-cultured with macrophages and the effect of increased expression of Siglec-E ligands analyzed. RESULTS: Siglec-E ligands are O-linked sialoglycoproteins with molecular weights of 70-300 kDa and are distributed broadly on mouse aorta as well as on MAECs in vitro. In vivo, the expression of Siglec-E ligands was increased in mice aortas in response to LPS treatment in an NF-κB signaling pathway dependent manner. In MAECs, the expression of Siglec-E ligands was also increased by LPS via an NF-κB signaling pathway. Deleted in malignant brain tumors-1 was identified to be one of multiple protein carriers of Siglec-E ligands, and glycans of ligands involved in MAECs induced by LPS. Notably, co-incubation of macrophages with LPS-treated MAECs induced macrophage apoptosis and decreased macrophage phagocytosis, effects that were completely reversed by blocking Siglec-E binding to Siglec-E ligands. CONCLUSIONS: These data demonstrated that Siglec-E ligands were highly expressed in response to LPS-induced vascular inflammation and inhibited the immune response of macrophages, which may be a therapeutic strategy to interfere with vascular inflammation.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Inmunosupresores/farmacología , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Escherichia coli/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Ligandos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Transducción de Señal/fisiología , Activación Transcripcional/efectos de los fármacosRESUMEN
AIM: Inflammation is a driving force in development of atherosclerosis, and hyperglycemia is a significant risk factor for angiopathy. Siglec-9, expressed on human neutrophils and macrophages, engages specific glycan ligands on tissues to diminish ongoing inflammation. MATERIALS AND METHOD: Siglec-9 ligands on human aorta were characterized and the effects of high glucose exposure on the expression of ligands for Siglec-9 on human umbilical vein endothelial cells (HUV-EC-C) in vitro and ligands for the comparable siglec (Siglec-E) on mouse aorta in vivo were studied. KEY FINDINGS: Siglec-9 ligands were expressed broadly on human aorta, as well as on HUV-EC-C. Siglec-9 ligands on HUV-EC-C were sharply up-regulated under high glucose exposure in vitro, as were Siglec-E ligands on the aortas of hyperglycemic mice. Exposure of HUV-EC-C to high-glucose resulted in consistent inhibitory changes in co-cultured macrophages including increased apoptosis and decreased phagocytosis. Control of Siglec-9 ligand expression on HUV-EC-C was downstream of changes in an enzyme involved in their biosynthesis, UDP-galactose-4-epimerase (GALE) and increased cellular N-acetylgalactosamine. The alteration of GALE was associated with the regulatory microRNA hsa-let-7f. SIGNIFICANCE: We conclude that exposure to high-glucose results in up-regulation of immune inhibitory Siglec-9 sialoglycan ligands on aorta and HUV-EC-C cells downstream of altered GALE and GalNAc expression, resulting in up-regulation of apoptosis and decrease of phagocytic activity of macrophages. Changes in Siglec-9 sialoglycan ligand expression on vascular endothelial cells may be a natural response to the initial steps of atherosclerosis and might be a potential target to regulate inflammation in diabetic angiopathy.