RESUMEN
The human mannose receptor expressed on macrophages and hepatic endothelial cells scavenges released lysosomal enzymes, glycopeptide fragments of collagen, and pathogenic microorganisms and thus reduces damage following tissue injury. The receptor binds mannose, fucose, or N-acetylglucosamine (GlcNAc) residues on these targets. C-type carbohydrate-recognition domain 4 (CRD4) of the receptor contains the site for Ca2+-dependent interaction with sugars. To investigate the details of CRD4 binding, glycan array screening was used to identify oligosaccharide ligands. The strongest signals were for glycans that contain either Manα1-2Man constituents or fucose in various linkages. The mechanisms of binding to monosaccharides and oligosaccharide substructures present in many of these ligands were examined in multiple crystal structures of CRD4. Binding of mannose residues to CRD4 results primarily from interaction of the equatorial 3- and 4-OH groups with a conserved principal Ca2+ common to almost all sugar-binding C-type CRDs. In the Manα1-2Man complex, supplementary interactions with the reducing mannose residue explain the enhanced affinity for this disaccharide. Bound GlcNAc also interacts with the principal Ca2+ through equatorial 3- and 4-OH groups, whereas fucose residues can bind in several orientations, through either the 2- and 3-OH groups or the 3- and 4-OH groups. Secondary contacts with additional sugars in fucose-containing oligosaccharides, such as the Lewis-a trisaccharide, provide enhanced affinity for these glycans. These results explain many of the biologically important interactions of the mannose receptor with both mammalian glycoproteins and microbes such as yeast and suggest additional classes of ligands that have not been previously identified.
Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Sitios de Unión , Carbohidratos/química , Carbohidratos/fisiología , Cristalografía por Rayos X/métodos , Disacáridos/metabolismo , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Lectinas Tipo C/fisiología , Ligandos , Manosa/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Lectinas de Unión a Manosa/fisiología , Glicoproteínas de Membrana/fisiología , Monosacáridos/metabolismo , Oligosacáridos/metabolismo , Polisacáridos/metabolismo , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos/fisiologíaRESUMEN
Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen-receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Lectinas Tipo C/metabolismo , Análisis por Matrices de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Bovinos , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Lectinas Tipo C/química , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcß1-2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the gene for CD23 in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic microorganisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.
Asunto(s)
Polisacáridos/metabolismo , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Bovinos , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores de IgE/químicaRESUMEN
The murine CLEC4f gene encodes the Kupffer cell receptor, a galactose-binding receptor containing a C-type carbohydrate-recognition domain. Orthologs have been identified in nearly 100 species. The receptors from rat and mouse have previously been characterized and data presented here show that functional CLEC4f protein is expressed in domestic cattle (Bos taurus). However, the human CLEC4f gene does not encode a functional receptor because a mutation in the splice acceptor site of the final exon prevents appropriate splicing and a missense mutation disrupts the sugar-binding site. Transcriptomic and PCR analysis of transcripts confirms the absence of a spliced transcript containing the final exon and only background levels of transcripts are detected in human tissues. These mutations are also present in the CLEC4f gene in Neanderthals. In contrast to humans, closely related species, including chimpanzees, do have CLEC4f genes that encode full-length receptors. Affinity chromatography and glycan array results demonstrate that the chimpanzee, bovine and murine proteins all bind to galactose, but they show preferences for different subsets of galactose-containing glycans. In non-human primates, the receptor is expressed in spleen rather than in liver. The results indicate that the CLEC4f protein probably has distinct functions in different species. Absence of the receptor precludes using it for targeting of glycoconjugates to cells in human liver. The fact that CLEC4f protein is expressed in spleen in non-human primates and the close evolutionary relationship of the CLEC4f protein to langerin (CD207) suggest that it may function in the immune system, possibly as a pathogen receptor.
Asunto(s)
Macrófagos del Hígado/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Animales , Humanos , Ratones , Receptores de Superficie Celular/deficienciaRESUMEN
Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1-2 or α1-4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1-2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man9GlcNAc2 oligosaccharide exhibited interaction with Manα1-2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1-2Man structures, whereas dectin-2 can bind Manα1-2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1-2Man disaccharide unit.
Asunto(s)
Disacáridos/metabolismo , Inmunidad Innata , Lectinas Tipo C/metabolismo , Manosa/metabolismo , Modelos Moleculares , Oligosacáridos/metabolismo , Polisacáridos/metabolismo , Sitios de Unión , Conformación de Carbohidratos , Cristalografía por Rayos X , Disacáridos/química , Epítopos/química , Epítopos/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Proteínas Inmovilizadas/metabolismo , Cuerpos de Inclusión/metabolismo , Cinética , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ligandos , Manosa/química , Oligosacáridos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Filogenia , Polisacáridos/química , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Blood dendritic cell antigen 2 (BDCA-2) is a C-type lectin found on the surface of plasmacytoid dendritic cells. It functions as a glycan-binding receptor that downregulates the production of type I interferons and thus plays a role in oligosaccharide-mediated immunomodulation. The carbohydrate recognition domain in BDCA-2 binds selectively to galactose-terminated bi-antennary glycans. Because the plasmacytoid dendritic cells function in a plasma environment rich in glycoproteins, experiments have been undertaken to identify endogenous ligands for blood dendritic cell antigen 2. A combination of blotting, affinity chromatography and proteomic analysis reveals that serum glycoprotein ligands for BDCA-2 include IgG, IgA and IgM. Compared to binding of IgG, which was previously described, IgA and IgM bind with higher affinity. The association constants for the different subclasses of immunoglobulins are below and roughly proportional to the serum concentrations of these glycoprotein ligands. Binding to the other main serum glycoprotein ligand, α2-macroglobulin, is independent of whether this protease inhibitor is activated. Binding to all of these glycoprotein ligands is mediated predominantly by bi-antennary glycans in which each branch bears a terminal galactose residue. The different affinities of the glycoprotein ligands reflect the different numbers of these galactose-terminated glycans and their degree of exposure on the native glycoproteins. The results suggest that normal serum levels of immunoglobulins could downmodulate interferon stimulation of further antibody production.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Galactosa/metabolismo , Glicoproteínas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Humanos , Ligandos , Unión ProteicaRESUMEN
The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages.
Asunto(s)
Lectinas Tipo C/química , Trehalosa/análogos & derivados , Trehalosa/química , Animales , Sitios de Unión , Calcio/química , Calcio/metabolismo , Bovinos , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Relación Estructura-Actividad , Trehalosa/metabolismoRESUMEN
Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galß1-3/4GlcNAcß1-2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca(2+) in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.
Asunto(s)
Galactosa/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Polisacáridos/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia de Carbohidratos , Cristalografía por Rayos X , Humanos , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Polisacáridos/química , Receptores Inmunológicos/genéticaRESUMEN
Mincle, the macrophage-inducible C-type lectin also known as CLEC-4E, binds to the mycobacterial glycolipid trehalose dimycolate and initiates a signaling cascade by serving as a receptor for Mycobacterium tuberculosis and other pathogenic mycobacterial species. Studies of the biological functions of human mincle often rely on mouse models, based on the assumption that the biological properties of the mouse receptor mimic those of the human protein. Experimental support for this assumption has been obtained by expression of the carbohydrate-recognition domain of mouse mincle and characterization of its interaction with small molecule analogs of trehalose dimycolate. The results confirm that the ligand-binding properties of mouse mincle closely parallel those of the human receptor. These findings are consistent with the conservation of key amino acid residues that have been shown to form the ligand-binding site in human and cow mincle. Sequence alignment reveals that these residues are conserved in a wide range of mammalian species, suggesting that mincle has a conserved function in binding ligands that may include endogenous mammalian glycans or pathogen glycans in addition to trehalose dimycolate.
Asunto(s)
Lectinas Tipo C/química , Proteínas de la Membrana/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Evolución Molecular , Expresión Génica , Humanos , Cinética , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes , Alineación de Secuencia , Trehalosa/metabolismoRESUMEN
Langerin, a C-type lectin on Langerhans cells, mediates carbohydrate-dependent uptake of pathogens in the first step of antigen presentation to the adaptive immune system. Langerin binds a diverse range of carbohydrates including high mannose structures, fucosylated blood group antigens, and glycans with terminal 6-sulfated galactose. Mutagenesis and quantitative binding assays indicate that salt bridges between the sulfate group and two lysine residues compensate for the nonoptimal binding of galactose at the primary Ca(2+) site. A commonly occurring single nucleotide polymorphism (SNP) in human langerin results in change of one of these lysine residues, Lys-313, to isoleucine. Glycan array screening reveals that this amino acid change abolishes binding to oligosaccharides with terminal 6SO4-Gal and enhances binding to oligosaccharides with terminal GlcNAc residues. Structural analysis shows that enhanced binding to GlcNAc may result from Ile-313 packing against the N-acetyl group. The K313I polymorphism is tightly linked to another SNP that results in the change N288D, which reduces affinity for glycan ligands by destabilizing the Ca(2+)-binding site. Langerin with Asp-288 and Ile-313 shows no binding to 6SO4-Gal-terminated glycans and increased binding to GlcNAc-terminated structures, but overall decreased binding to glycans. Altered langerin function in individuals with the linked N288D and K313I polymorphisms may affect susceptibility to infection by microorganisms.
Asunto(s)
Sustitución de Aminoácidos , Antígenos CD/química , Calcio/química , Lectinas Tipo C/química , Lectinas de Unión a Manosa/química , Oligosacáridos/química , Polimorfismo de Nucleótido Simple , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Sitios de Unión , Calcio/inmunología , Calcio/metabolismo , Cristalografía por Rayos X , Predisposición Genética a la Enfermedad/genética , Humanos , Infecciones/genética , Infecciones/inmunología , Infecciones/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ligandos , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/inmunología , Lectinas de Unión a Manosa/metabolismo , Oligosacáridos/genética , Oligosacáridos/inmunología , Oligosacáridos/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Terciaria de ProteínaRESUMEN
Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1-1Glcα headgroup is liganded to a Ca(2+) in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.
Asunto(s)
Glucolípidos/metabolismo , Lectinas Tipo C/metabolismo , Mycobacterium/metabolismo , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas Tipo C/química , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores Inmunológicos/química , Trehalosa/química , Trehalosa/metabolismoRESUMEN
Trehalose dimycolate, an unusual glycolipid in the outer membrane of Mycobacterium tuberculosis, stimulates macrophages by binding to the macrophage receptor mincle. This stimulation plays an important role both in infection by mycobacteria and in the use of derivatives of mycobacteria as adjuvants to enhance the immune response. The mechanism of trehalose dimycolate binding to the C-type carbohydrate-recognition domain in human mincle has been investigated using a series of synthetic analogs of trehalose dimycolate and site-directed mutagenesis of the human protein. The results support a mechanism of binding acylated trehalose derivatives to human mincle that is very similar to the mechanism of binding to bovine mincle, in which one glucose residue in the trehalose headgroup of the glycolipid is ligated to the principle Ca(2+)-binding site in the carbohydrate-recognition domain, with specificity for the disaccharide resulting from interactions with the second glucose residue. Acyl chains attached to the 6-OH groups of trehalose enhance affinity, with the affinity dependent on the length of the acyl chains and the presence of a hydrophobic groove adjacent to the sugar-binding sites. The results indicate that the available crystal structure of the carbohydrate-recognition domain of human mincle is unlikely to be in a fully active conformation. Instead, the ligand-binding conformation probably resembles closely the structure observed for bovine mincle in complex with trehalose. These studies provide a basis for targeting human mincle as a means of inhibiting interactions with mycobacteria and as an approach to harnessing the ability of mincle to stimulate the immune response.
Asunto(s)
Factores Cordón/química , Lectinas Tipo C/química , Mycobacterium tuberculosis/química , Receptores Inmunológicos/química , Animales , Sitios de Unión , Calcio/química , Bovinos , Factores Cordón/síntesis química , Humanos , Concentración de Iones de Hidrógeno , Lectinas Tipo C/metabolismo , Modelos Moleculares , Conformación Proteica , Receptores Inmunológicos/metabolismoRESUMEN
Diverse glycans found on the surfaces of mammalian cells provide a basis for selective adhesion between cells mediated by glycan-specific receptors. Well-understood examples of cell adhesion based on such interactions include selectin-mediated rolling of leukocytes on endothelia. Other receptors with similar selectivity for specific sugar epitopes on cell surfaces are being characterised. However, the simple paradigm of adhesion resulting from receptors on one cell binding to glycans on another cell applies in only a limited number of systems. Instead, glycans and receptor-glycan interactions often modulate adhesion in indirect ways, such as by changing the organisation of cell surface glycoproteins and by antagonising the effect of protein adhesion systems.
Asunto(s)
Adhesión Celular/fisiología , Polisacáridos , Receptores de Superficie Celular/metabolismo , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Colectinas/química , Colectinas/metabolismo , Galectinas/química , Galectinas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Antígeno Lewis X/química , Antígeno Lewis X/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Receptores de Superficie Celular/química , Receptores Depuradores/química , Receptores Depuradores/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismoRESUMEN
The properties of the human macrophage galactose receptor have been investigated. Specificity for N-acetylgalactosamine (GalNAc) residues with exposed 3- and 4-hydroxyl groups explains virtually all of the results obtained from a recently expanded array of synthetic glycans and is consistent with a model for the structure of the binding site. This simple interaction is sufficient to explain the ability of the receptor to bind to tumor-cell glycans bearing Tn and sialyl-Tn antigens, but not to more elaborate O-linked glycans that predominate on normal cells. This specificity also allows for binding of parasite glycans and screening of an array of bacterial outer membrane oligosaccharides confirms that the receptor binds to a subset of these structures with appropriately exposed GalNAc residues. A key feature of the receptor is the clustering of binding sites in the extracellular portion of the protein, which retains the trimeric structure observed in the cell membrane. Chemical crosslinking, gel filtration, circular dichroism analysis and differential scanning calorimetry demonstrate that this trimeric structure of the receptor is stabilized by an α-helical coiled coil that extends from the surface of the membrane to the globular carbohydrate-recognition domains. The helical neck domains form independent trimerization domains. Taken together, these results indicate that the macrophage galactose receptor shares many of the features of serum mannose-binding protein, in which clusters of monosaccharide-binding sites serve as detectors for a simple epitope that is not common on endogenous cell surface glycans but that is abundant on the surfaces of tumor cells and certain pathogens.
Asunto(s)
Acetilglucosamina/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Acetilglucosamina/química , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Sitios de Unión , Humanos , Macrófagos/química , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismoRESUMEN
The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewis(x) groups (Galß1-4(Fucα1-3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewis(x) groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewis(x) groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.
Asunto(s)
Colectinas/metabolismo , Glicoproteínas/metabolismo , Neutrófilos/metabolismo , Receptores Depuradores/metabolismo , Vesículas Secretoras/metabolismo , Trisacáridos/metabolismo , Colectinas/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fucosa/genética , Fucosa/metabolismo , Regulación de la Expresión Génica/fisiología , Glicoproteínas/genética , Humanos , Antígeno Lewis X/análogos & derivados , Ligandos , Neutrófilos/citología , Especificidad de Órganos/fisiología , Unión Proteica , Receptores Depuradores/genética , Vesículas Secretoras/genética , Trisacáridos/genéticaRESUMEN
Genital herpes is the most prevalent viral sexually transmitted infection worldwide and is mainly caused by HSV type 2 (HSV-2). HSV-2 infection enhances HIV-1 susceptibility, even in the absence of clinical symptoms. In this study, we investigated the effect of HSV-2 on HIV-1 transmission by mucosal Langerhans cells (LCs). LCs are important in heterosexual transmission because they form a barrier against HIV-1 infection; LCs efficiently capture and degrade HIV-1 through the C-type lectin langerin, thereby preventing HIV-1 transmission. Notably, our data showed that HSV-2 enhanced HIV-1 infection of LCs and subsequent HIV-1 transmission to T cells. HSV-2 interfered with HIV-1 capture by langerin, which allowed efficient HIV-1 infection of LCs. HSV-2 inhibited the antiviral function of langerin at two levels; HSV-2 decreased langerin expression and competed with HIV-1 for langerin binding. HSV-2 replication was not required, because both UV-inactivated HSV-2 and TLR-3 agonist polyinosinic:polycytidylic acid similarly increased HIV-1 transmission by LCs. Therefore, we identified a mechanism by which HSV-2 enhances HIV-1 susceptibility, even in the absence of clinical symptoms. Our data demonstrated that viral coinfections, such as HSV-2, breach the protective function of LCs by abrogating langerin function, which increases HIV-1 susceptibility. These data reinforce the importance of preventing sexually transmitted infections, such as HSV-2, to reduce the transmission of HIV-1.
Asunto(s)
VIH-1/inmunología , Herpesvirus Humano 2/inmunología , Células de Langerhans/inmunología , Células de Langerhans/virología , Antígenos CD/biosíntesis , Antígenos CD/metabolismo , Unión Competitiva/inmunología , Línea Celular , Susceptibilidad a Enfermedades/inmunología , Infecciones por VIH/complicaciones , Infecciones por VIH/patología , Infecciones por VIH/transmisión , VIH-1/metabolismo , Herpes Genital/complicaciones , Herpes Genital/patología , Herpesvirus Humano 2/crecimiento & desarrollo , Herpesvirus Humano 2/metabolismo , Humanos , Células Jurkat , Células de Langerhans/patología , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/antagonistas & inhibidores , Lectinas de Unión a Manosa/biosíntesis , Lectinas de Unión a Manosa/metabolismo , Factores de Riesgo , Virión/metabolismo , Activación Viral/inmunologíaRESUMEN
The dendritic cell receptor DC-SIGN mediates pathogen recognition by binding to glycans characteristic of pathogen surfaces, including those found on HIV. Clustering of carbohydrate-binding sites in the receptor tetramer is believed to be critical for targeting of pathogen glycans, but the arrangement of these sites remains poorly understood. Surface force measurements between apposed lipid bilayers displaying the extracellular domain of DC-SIGN and a neoglycolipid bearing an oligosaccharide ligand provide evidence that the receptor is in an extended conformation and that glycan docking is associated with a conformational change that repositions the carbohydrate-recognition domains during ligand binding. The results further show that the lateral mobility of membrane-bound ligands enhances the engagement of multiple carbohydrate-recognition domains in the receptor oligomer with appropriately spaced ligands. These studies highlight differences between pathogen targeting by DC-SIGN and receptors in which binding sites at fixed spacing bind to simple molecular patterns.
Asunto(s)
Sitios de Unión/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Adhesividad , Células Dendríticas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Oligosacáridos/metabolismo , Conformación ProteicaRESUMEN
Force-distance measurements have been used to examine differences in the interaction of the dendritic cell glycan-binding receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR (L-SIGN) with membranes bearing glycan ligands. The results demonstrate that upon binding to membrane-anchored ligand, DC-SIGNR undergoes a conformational change similar to that previously observed for DC-SIGN. The results also validate a model for the extracellular domain of DC-SIGNR derived from crystallographic studies. Force measurements were performed with DC-SIGNR variants that differ in the length of the neck that result from genetic polymorphisms, which encode different numbers of the 23-amino acid repeat sequences that constitute the neck. The findings are consistent with an elongated, relatively rigid structure of the neck repeat observed in crystals. In addition, differences in the lengths of DC-SIGN and DC-SIGNR extracellular domains with equivalent numbers of neck repeats support a model in which the different dispositions of the carbohydrate-recognition domains in DC-SIGN and DC-SIGNR result from variations in the sequences of the necks.
Asunto(s)
Moléculas de Adhesión Celular/química , Espacio Extracelular/química , Lectinas Tipo C/química , Conformación Proteica , Mapeo de Interacción de Proteínas/métodos , Receptores de Superficie Celular/química , Secuencias de Aminoácidos , Moléculas de Adhesión Celular/metabolismo , Elasticidad , Espacio Extracelular/metabolismo , Humanos , Interferometría/métodos , Lectinas Tipo C/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Secuencias Repetitivas de Aminoácido , Resonancia por Plasmón de Superficie/métodos , Propiedades de SuperficieRESUMEN
Langerin, an endocytic receptor of Langerhans cells, binds pathogens such as human immunodeficiency virus by recognition of surface glycoconjugates and mediates their internalization into Birbeck granules. Langerin has an extracellular region consisting of a C-type carbohydrate-recognition domain (CRD) and a neck region that stabilizes formation of trimers. As in many other C-type lectins, oligomerization is required for high affinity binding to glycan ligands and is also likely to be important for determining specificity. To facilitate structural analysis of the human langerin trimer, a truncated form of the extracellular region, consisting of part of the neck and the CRD, has been characterized. Like the full-length protein, truncated langerin exists as a stable trimer in solution. Glycan array screening with the trimeric fragment shows that high mannose oligosaccharides are the best ligands for langerin. Structural analysis of the trimeric fragment of langerin confirms that the neck region forms a coiled-coil of alpha-helices. Multiple interactions between the neck region and the CRDs make the trimer a rigid unit with the three CRDs in fixed positions and the primary sugar-binding sites separated by a distance of 42 A. The fixed orientation of the sugar-binding sites in the trimer is likely to place constraints on the ligands that can be bound by langerin.
Asunto(s)
Antígenos CD/química , Lectinas Tipo C/química , Lectinas de Unión a Manosa/química , Manosa/química , Oligosacáridos/química , Multimerización de Proteína , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Manosa/metabolismo , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Oligosacáridos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcß1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcß1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.