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
Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE-FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow-derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has no protection in mice that do not express Siglec-8. Furthermore, mice protected from anaphylaxis remain desensitized to subsequent allergen challenge because of loss of Ag-specific IgE from the cell surface and accelerated clearance of IgE from the blood. Thus, although expression of human Siglec-8 on murine mast cells does not by itself modulate IgE-FcεRI-mediated cell activation, the enforced recruitment of Siglec-8 to the FcεRI receptor by Sig8L-decorated antigenic liposomes results in inhibition of degranulation and desensitization to subsequent Ag exposure.
Asunto(s)
Alérgenos/administración & dosificación , Anafilaxia/tratamiento farmacológico , Anafilaxia/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Desensibilización Inmunológica/métodos , Sistemas de Liberación de Medicamentos/métodos , Inmunoglobulina E/metabolismo , Lectinas/metabolismo , Mastocitos/inmunología , Nanopartículas/química , Polisacáridos/administración & dosificación , Receptores de IgE/metabolismo , Anafilaxia/inmunología , Animales , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos B/genética , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Lectinas/genética , Ligandos , Liposomas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Polisacáridos/metabolismo , Ratas , Receptores de IgE/genética , Resultado del TratamientoRESUMEN
CD22 and sialic acid-binding Ig-like lectin (Siglec)-G are members of the Siglec family of inhibitory coreceptors expressed on B cells that participate in enforcement of peripheral B cell tolerance. We have shown previously that when a BCR engages its cognate Ag on a cell surface that also expresses Siglec ligands, B cell Siglecs are recruited to the immunological synapse, resulting in suppression of BCR signaling and B cell apoptosis. Because all cells display sialic acids, and CD22 and Siglec-G have distinct, yet overlapping, specificities for sialic acid-containing glycan ligands, any cell could, in principle, invoke this tolerogenic mechanism for cell surface Ags. However, we show in this article that C57BL/6J mouse RBCs are essentially devoid of CD22 and Siglec-G ligands. As a consequence, RBCs that display a cell surface Ag, membrane-bound hen egg lysozyme, strongly activate Ag-specific B cells. We reasoned that de novo introduction of CD22 ligands in RBCs should abolish B cell activation toward its cognate Ag on the surface of RBCs. Accordingly, we used a glyco-engineering approach wherein synthetic CD22 ligands linked to lipids are inserted into the membrane of RBCs. Indeed, insertion of CD22 ligands into the RBC cell surface strongly inhibited B cell activation, cytokine secretion, and proliferation. These results demonstrate that the lack of Siglec ligands on the surface of murine RBCs permits B cell responses to erythrocyte Ags and show that Siglec-mediated B cell tolerance is restricted to cell types that express glycan ligands for the B cell Siglecs.
Asunto(s)
Antígenos de Superficie/inmunología , Linfocitos B/inmunología , Eritrocitos/inmunología , Tolerancia Inmunológica/inmunología , Activación de Linfocitos/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/metabolismo , Células CHO , Línea Celular , Cricetulus , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Muramidasa/genética , Muramidasa/inmunología , Muramidasa/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Ácidos Siálicos/inmunologíaRESUMEN
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
The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Sustitución de Aminoácidos , Antígenos Virales/química , Antígenos Virales/inmunología , HumanosRESUMEN
Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.
Asunto(s)
Polisacáridos/química , Células HeLa , Humanos , Liposomas/química , Liposomas/metabolismo , Polisacáridos/metabolismoRESUMEN
Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avian receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted.IMPORTANCE In the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can aid in global surveillance of such viruses for potential spread and emerging threat to the human population.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/química , Animales , Australia , Sitios de Unión , Aves/virología , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H7N9 del Virus de la Influenza A , Virus de la Influenza A/clasificación , Virus de la Influenza A/metabolismo , Gripe Humana/virología , Modelos Moleculares , Polisacáridos , Unión Proteica , Conformación Proteica , Receptores Virales/metabolismo , SiberiaRESUMEN
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
Unwanted antibody responses significantly impact human health, and current options for treating deleterious antibody responses largely rely on broad immunosuppressants that can compromise overall immunity. A desirable alternative is to induce antigen-specific immune tolerance. We have shown that co-presentation of antigen and ligands of Bâ cell sialic acid-binding immunoglobulin-like lectins (Siglecs) on a liposomal nanoparticle induces antigen-specific tolerance. Although Siglec-engaging tolerance-inducing antigenic liposomes (STALs) induce robust B cell tolerance in naïve mice, the full potential of STALs requires long-term tolerance induction and suppression of an ongoing immune response. We hypothesized that STALs encapsulated with rapamycin (RAPA), an immunomodulator, could improve the efficacy of STALs and potentially enable their use in the context of immunological memory. Here, we showed that formulation of STALs with RAPA produced enhanced tolerance induction in naïve mice compared to STALs without RAPA but had minimal impact on inducing tolerance in previously sensitized mice. These findings indicate that the addition of immunomodulators to STALs could be beneficial in tolerance induction and support future development of STALs for the treatment of allergy and autoimmune diseases.
Asunto(s)
Antialérgicos/farmacología , Hipersensibilidad al Huevo/terapia , Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Liposomas/farmacología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Sirolimus/farmacología , Animales , Antialérgicos/inmunología , Anticuerpos/sangre , Anticuerpos/efectos de los fármacos , Antígenos/inmunología , Antígenos/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Composición de Medicamentos , Hipersensibilidad al Huevo/genética , Hipersensibilidad al Huevo/inmunología , Expresión Génica , Humanos , Inmunosupresores/química , Ligandos , Liposomas/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Sirolimus/químicaRESUMEN
Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169(+) macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169(+) macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169(-/-) mice and is macrophage-dependent, demonstrating that targeting CD169(+) macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte-derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells.
Asunto(s)
Lípidos/inmunología , Macrófagos/metabolismo , Células T Asesinas Naturales/citología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Animales , Presentación de Antígeno , Antígenos/inmunología , Línea Celular , Células Dendríticas/citología , Endocitosis , Glucolípidos/inmunología , Humanos , Ligandos , Liposomas/metabolismo , Hígado/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Monocitos/citologíaRESUMEN
Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A , Gripe Humana , Animales , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Receptores Virales/química , Ácidos Siálicos/metabolismo , Polisacáridos/metabolismo , Pollos , Glicoproteínas Hemaglutininas del Virus de la InfluenzaRESUMEN
Sialosides on N- and O-linked glycoproteins play a fundamental role in many biological processes, and synthetic glycan probes have proven to be valuable tools for elucidating these functions. Though sialic acids are typically found α2-3- or α2-6-linked to a terminal nonreducing end galactose, poly-LacNAc extended core-3 O-linked glycans isolated from rat salivary glands and human colonic mucins have been reported to contain multiple internal Neu5Acα2-6Gal epitopes. Here, we have developed an efficient approach for the synthesis of a library of N- and O-linked glycans with multisialylated poly-LacNAc extensions, including naturally occurring multisialylated core-3 O-linked glycans. We have found that a recombinant α2-6 sialyltransferase from Photobacterium damsela (Pd2,6ST) exhibits unique regioselectivity and is able to sialylate internal galactose residues in poly-LacNAc extended glycans which was confirmed by MS/MS analysis. Using a glycan microarray displaying this library, we found that Neu5Acα2-6Gal specific influenza virus hemagglutinins, siglecs, and plant lectins are largely unaffected by adjacent internal sialylation, and in several cases the internal sialic acids are recognized as ligands. Polyclonal IgY antibodies specific for internal sialoside epitopes were elicited in inoculated chickens.
Asunto(s)
Photobacterium/enzimología , Polisacáridos/síntesis química , Ácidos Siálicos/química , Sialiltransferasas/química , Humanos , Polisacáridos/química , Polisacáridos/farmacologíaRESUMEN
The 2009 H1N1 influenza pandemic is the first human pandemic in decades and was of swine origin. Although swine are believed to be an intermediate host in the emergence of new human influenza viruses, there is still little known about the host barriers that keep swine influenza viruses from entering the human population. We surveyed swine progenitors and human viruses from the 2009 pandemic and measured the activities of the hemagglutinin (HA) and neuraminidase (NA), which are the two viral surface proteins that interact with host glycan receptors. A functional balance of these two activities (HA binding and NA cleavage) is found in human viruses but not in the swine progenitors. The human 2009 H1N1 pandemic virus exhibited both low HA avidity for glycan receptors as a result of mutations near the receptor binding site and weak NA enzymatic activity. Thus, a functional match between the hemagglutinin and neuraminidase appears to be necessary for efficient transmission between humans and may be an indicator of the pandemic potential of zoonotic viruses.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Gripe Humana/virología , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/veterinaria , Enfermedades de los Porcinos/virología , Proteínas Virales/metabolismo , Animales , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Gripe Humana/metabolismo , Cinética , Neuraminidasa/química , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Pandemias , Polisacáridos/química , Polisacáridos/metabolismo , Unión Proteica , Receptores Virales/química , Receptores Virales/metabolismo , Porcinos , Enfermedades de los Porcinos/metabolismo , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3' sialyllactosamine (3'-SLN) and 6'-SLN sialosides with equilibrium dissociation constant (K(D)) values of 30.0 µM and 645 µM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/enzimología , Neuraminidasa/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Humanos , Modelos Moleculares , Mutación , Neuraminidasa/química , Neuraminidasa/genética , Unión Proteica , Proteolisis , Especificidad por SustratoRESUMEN
Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Receptores Virales/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Gripe Humana/metabolismo , Gripe Humana/virología , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Pandemias , Polisacáridos/química , Polisacáridos/metabolismo , Unión Proteica , Receptores Virales/metabolismo , Especificidad de la EspecieRESUMEN
Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.
RESUMEN
Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galß1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid.
Asunto(s)
Adenovirus Caninos/genética , Proteínas de la Cápside/metabolismo , Polisacáridos/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ácidos Siálicos/metabolismo , Animales , Biomarcadores/metabolismo , Proteínas de la Cápside/genética , Perros , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genéticaRESUMEN
Cell-specific delivery of therapeutic agents using ligand targeting is gaining interest because of its potential for increased efficacy and reduced side effects. The challenge is to develop a suitable ligand for a cell-surface receptor that is selectively expressed on the desired cell. Sialoadhesin (Sn, Siglec-1, CD169), a sialic acid-binding immunoglobulin-like lectin (Siglec) expressed on subsets of resident and inflammatory macrophages, is an attractive target for the development of a ligand-targeted delivery system. Here we report the development of a high-affinity and selective ligand for Sn that is an analogue of the natural ligand and is capable of targeting liposomal nanoparticles to Sn-expressing cells in vivo. An efficient in silico screen of a library of â¼8400 carboxylic acids was the key to identifying novel 9-N-acyl-substituted N-acetylneuramic acid (Neu5Ac) substituents as potential lead compounds. A small panel of targets were selected from the screen and synthesized to evaluate their affinities and selectivities. The most potent of these Sn ligands, 9-N-(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Acα2-3Galß1-4GlcNAc ((TCC)Neu5Ac), was conjugated to lipids for display on a liposomal nanoparticle for evaluation of targeted delivery to cells. The (TCC)Neu5Ac liposomes were found to target liposomes selectively to cells expressing either murine or human Sn in vitro, and when administered to mice, they exhibited in vivo targeting to Sn-positive macrophages.
Asunto(s)
Adhesión Celular , Macrófagos/efectos de los fármacos , Ácido N-Acetilneuramínico/química , Polisacáridos/química , Animales , Separación Celular , Citometría de Flujo , Ligandos , RatonesRESUMEN
Human influenza viruses are proposed to recognize sialic acids (pink diamonds) on glycans extended with poly-LacNAc chains (LacNAc=(yellow circle+blue square)). N- and O-linked glycans were extended with different poly-LacNAc chains with α2-3- and α2-6-linked sialic acids recognized by human and avian influenza viruses, respectively. The specificity of recombinant hemagglutinins (receptors in green) was investigated by using glycan microarray technology.
Asunto(s)
Hemaglutininas/metabolismo , Virus de la Influenza A/metabolismo , Gripe Aviar/virología , Gripe Humana/virología , Polisacáridos/metabolismo , Ácidos Siálicos/metabolismo , Animales , Aves , Secuencia de Carbohidratos , Hemaglutininas/química , Humanos , Virus de la Influenza A/química , Análisis por Micromatrices , Datos de Secuencia Molecular , Polisacáridos/química , Ácidos Siálicos/químicaRESUMEN
Myelin associated glycoprotein (Siglec-4) is a myelin adhesion receptor, that is, well established for its role as an inhibitor of axonal outgrowth in nerve injury, mediated by binding to sialic acid containing ligands on the axonal membrane. Because disruption of myelin-ligand interactions promotes axon outgrowth, we have sought to develop potent ligand based inhibitors using natural ligands as scaffolds. Although natural ligands of MAG are glycolipids terminating in the sequence NeuAcα2-3Galß1-3(±NeuAcα2-6)GalNAcß-R, we previously established that synthetic O-linked glycoprotein glycans with the same sequence α-linked to Thr exhibited â¼1000-fold increased affinity (â¼1µM). Attempts to increase potency by introducing a benzoylamide substituent at C-9 of the α2-3 sialic acid afforded only a two-fold increase, instead of increases of >100-fold observed for other sialoside ligands of MAG. Surprisingly, however, introduction of a 9-N-fluoro-benzoyl substituent on the α2-6 sialic acid increased affinity 80-fold, resulting in a potent inhibitor with a K(d) of 15nM. Docking this ligand to a model of MAG based on known crystal structures of other siglecs suggests that the Thr positions the glycan such that aryl substitution of the α2-3 sialic acid produces a steric clash with the GalNAc, while attaching an aryl substituent to the other sialic acid positions the substituent near a hydrophobic pocket that accounts to the increase in affinity.