RESUMO
Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Galinhas , Evolução Molecular , Furões , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/transmissão , Influenza Humana/virologia , Mutação , Neuraminidase/genética , Análise de Sequência com Séries de Oligonucleotídeos , Vírus Reordenados/genética , Vírus Reordenados/fisiologia , Receptores Virais/genética , Análise de Sequência de RNAAssuntos
Antígenos/biossíntese , Células CHO/metabolismo , Dissacarídeos/biossíntese , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Domínio Catalítico , Cricetinae , Cricetulus , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells. Surprisingly, anti-NP antibodies of lower valency, IgA (n = 4) and IgG (n = 2), were also found to drive complex formation, though with lower avidity. Ligands bearing alternate linkers of variable length and structure were constructed to establish the importance of a minimal length requirement, and versatility in the structural requirement. We show that the ligand drives assembly of IgM complexes exclusively on the surface of B cells and not other classes of white blood cells that do not express CD22, which lends itself to the possibility of targeting B cells in certain hematopoietic malignancies.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Linfócitos B/imunologia , Imunoglobulina M/imunologia , Oligossacarídeos/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Cricetinae , Cricetulus , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Ligantes , Camundongos , Nitrofenóis/imunologiaRESUMO
CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia alpha2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Endocitose , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Açúcares Ácidos/metabolismo , Animais , Linfócitos B/imunologia , Morte Celular , Células Cultivadas , Humanos , Ligantes , Camundongos , Estrutura Molecular , Açúcares Ácidos/químicaRESUMO
The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation.
Assuntos
Linfócitos B/imunologia , Endocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptores Fc/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Sialiltransferases/metabolismo , Animais , Linfócitos B/química , Linfócitos B/metabolismo , Endocitose/genética , Glicoproteínas/metabolismo , Glicosilação , Imunidade/genética , Imunoglobulina M/análise , Imunoglobulina M/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/análise , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Microdomínios da Membrana/química , Camundongos , Camundongos Mutantes , Ácido N-Acetilneuramínico/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/análise , Receptores de Antígenos de Linfócitos B/análise , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Fc/análise , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/análise , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Sialiltransferases/genética , Transdução de Sinais , Tirosina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Information contained in the mammalian glycome is decoded by glycan-binding proteins (GBPs) that mediate diverse functions including host-pathogen interactions, cell trafficking and transmembrane signaling. Although information on the biological roles of GBPs is rapidly expanding, challenges remain in identifying the glycan ligands and their impact on GBP function. Protein-glycan interactions are typically low affinity, requiring multivalent interactions to achieve a biological effect. Though many glycoproteins can carry the glycan structure recognized by the GBP, other factors, such as recognition of protein epitopes and microdomain localization, may restrict which glycoproteins are functional ligands in situ. Recent advances in development of glycan arrays, synthesis of multivalent glycan ligands, bioengineering of cell-surface glycans and glycomics databases are providing new tools to identify the ligands of GBPs and to elucidate the mechanisms by which they participate in GBP function.
Assuntos
Engenharia Biomédica , Microdomínios da Membrana/metabolismo , Polissacarídeos/metabolismo , Proteoma , Configuração de Carboidratos , Sequência de Carboidratos , Bases de Dados Factuais , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Ligantes , Análise em Microsséries , Dados de Sequência Molecular , Polissacarídeos/química , Polissacarídeos/genética , Ligação Proteica , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
CD22 is a negative regulator of B cell signaling, an activity modulated by its interaction with glycan ligands containing alpha2-6-linked sialic acids. B cells deficient in the enzyme (ST6Gal I) that forms the CD22 ligand show suppressed BCR signaling. Here we report that mice deficient in both CD22 and its ligand (Cd22-/- St6gal1-/- mice) showed restored B cell receptor (BCR) signaling, suggesting that the suppressed signaling of St6gal1-/- cells is mediated through CD22. Coincident with suppressed BCR signaling, B cells lacking ST6Gal I showed a net redistribution of the BCR to clathrin-rich microdomains containing most of the CD22, resulting in a twofold increase in the localization of CD22 together with the BCR. These studies suggest an important function for the CD22-ligand interaction in regulating BCR signaling and microdomain localization.
Assuntos
Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Linfócitos B/enzimologia , Vesículas Revestidas por Clatrina/imunologia , Técnicas In Vitro , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Sialiltransferases/deficiência , Sialiltransferases/genética , Sialiltransferases/metabolismo , Transdução de Sinais , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
CD22 is a negative regulator of B-cell receptor signaling, an activity mediated by recruitment of SH2 domain-containing phosphatase 1 through a phosphorylated immunoreceptor tyrosine inhibitory motif in its cytoplasmic domain. As in other members of the sialic acid-binding immunoglobulin-like lectin, or siglec, family, the extracellular N-terminal immunoglobulin domain of CD22 binds to glycan ligands containing sialic acid, which are highly expressed on B-cell glycoproteins. B-cell glycoproteins bind to CD22 in cis and 'mask' the ligand-binding domain, modulating its activity as a regulator of B-cell signaling. To assess cell-surface cis ligand interactions, we developed a new method for in situ photoaffinity cross-linking of glycan ligands to CD22. Notably, CD45, surfaceIgM (sIgM) and other glycoproteins that bind to CD22 in vitro do not appear to be important cis ligands of CD22 in situ. Instead, CD22 seems to recognize glycans of neighboring CD22 molecules as cis ligands, forming homomultimeric complexes.
Assuntos
Linfócitos B/metabolismo , Polissacarídeos/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/química , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Azidas/síntese química , Azidas/química , Azidas/farmacologia , Linhagem Celular , Estrutura Molecular , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Polissacarídeos/química , Ligação Proteica , Estrutura Quaternária de Proteína , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genéticaRESUMO
Glycan-binding proteins mediate diverse aspects of cell biology including pathogen recognition of host cells, cell trafficking, endocytosis and modulation of cell signaling. This is accomplished despite the intrinsic low affinity for their ligands through multivalent interactions that increase effective affinity and adhesive force. Recent successes in the rational design of high-affinity ligands for glycan-binding proteins offer the promise to create well-defined tools for exploring the structure and functions of this class of receptors.
Assuntos
Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Adesão Celular , Comunicação Celular , Movimento Celular , Ligantes , Ligação Proteica , Proteínas Virais/metabolismoRESUMO
Following activation in the periphery, murine CD8+ T cells exhibit a characteristic increased binding of peanut agglutinin (PNA), reflecting an increased expression of hyposialylated O-linked glycans (Galbeta1-3GalNAcalpha-O-Thr/Ser) on the cell surface. In this report, we show that the majority of the PNA receptors expressed on activated CD8+ T cells are carried by CD45. Other glycoproteins (e.g. CD8) and the glycolipid asialo-GM1 also carry PNA receptors, although to a much lesser extent. Analysis of enzymes involved in the sialylation/de-sialylation pathways showed that generation of PNA receptors in activated CD8+ T cells is not due to up-regulation of endogenous sialidases. Instead, our results indicate that the PNA(high) phenotype results from de novo synthesis of CD45 carrying reduced sialylated core 1 O-glycans.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Antígenos Comuns de Leucócito/biossíntese , Aglutinina de Amendoim/metabolismo , Polissacarídeos/metabolismo , Animais , Biotina/química , Biotina/metabolismo , Western Blotting , Células COS , Membrana Celular/metabolismo , Cromatografia em Camada Fina , Citometria de Fluxo , Glicoproteínas/química , Lectinas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Químicos , Neuraminidase/metabolismo , Fenótipo , Polissacarídeos/química , Testes de Precipitina , Ligação Proteica , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialiltransferases/química , Baço/citologia , Linfócitos T/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
CD22, a negative regulator of B cell signaling, is a member of the siglec family that binds to alpha2-6-linked sialic acids on glycoproteins. Previous reports demonstrated that binding of multivalent sialoside probes to CD22 is blocked, or "masked," by endogenous (cis) ligands, unless they are first destroyed by sialidase treatment. These results suggest that cis ligands on B cells make CD22 functionally unavailable for binding to ligands in trans. Through immunofluorescence microscopy, however, we observed that CD22 on resting B cells redistributes to the site of contact with other B or T lymphocytes. Redistribution is mediated by interaction with trans ligands on the opposing cell because it does not occur with ligand-deficient lymphocytes from ST6GalI-null mice. Surprisingly, CD45, proposed as both a cis and trans ligand of CD22, was not required for redistribution to sites of cell contact, given that redistribution of CD22 was independent of CD45 and was observed with lymphocytes from CD45-deficient mice. Furthermore, CD45 is not required for CD22 masking as similar levels of masking were observed in the WT and null mice. Comparison of the widely used sialoside-polyacrylamide probe with a sialoside-streptavidin probe revealed that the latter bound a subset of B cells without sialidase treatment, suggesting that cis ligands differentially impacted the binding of these two probes in trans. The combined results suggest that equilibrium binding to cis ligands does not preclude binding of CD22 to ligands in trans, and allows for its redistribution to sites of contact between lymphocytes.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Moléculas de Adesão Celular , Lectinas/metabolismo , Animais , Citometria de Fluxo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Sondas Moleculares , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
CD22, a B cell-specific member of the Siglec family, is an important inhibitor of B cell signaling. The first Ig-like domain of CD22 specifically binds to alpha2,6-linked sialic acids. Through these interactions CD22 can mediate adhesion to other cells in trans, but can also bind endogenous ligands on the B cell surface in cis. Cis binding of CD22 to sialylated ligands enhances the efficiency of inhibition and thereby reduces the BCR signaling strength. In this study we used a newly developed oligomeric streptavidin-based sialylated probe as an artificial CD22 ligand. We found that CD22 is bound to ligands in cis on most B cells. However, there is a proportion of B cells with unbound (unmasked) CD22. The subpopulation with unmasked CD22 is 2-fold increased in transitional and marginal zone B cells in the spleen and on B1 cells in the peritoneum, when compared to mature B cells. Also, B cells with unmasked CD22 have an activated phenotype. Unmasking of CD22 could be functionally involved in lowering the signaling threshold on developmental checkpoints such as transitional B cells and during B cell activation or could be a consequence of such activation processes.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/imunologia , Moléculas de Adesão Celular , Lectinas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos B/análise , Subpopulações de Linfócitos B/química , Lectinas/análise , Ativação Linfocitária , Camundongos , Sondas Moleculares/química , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Baço/citologia , Baço/imunologiaRESUMO
Systematic synthesis and myelin-associated glycoprotein (MAG)-binding activity of novel sulfated GM1b analogues structurally related to Chol-1 (alpha-series) gangliosides, high-affinity ligands for neural siglecs, are described. The suitably protected gangliotriose derivatives, 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside and 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,6-di-O-benzyl-3-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside were each glycosylated with alpha-NeuAc-(2-->3)-galactose donor to give the corresponding pentasaccharides in 94% (beta1,3 glycoside only) and 90% (beta1,3:beta1,4 = 2:1), respectively. After proper manipulation of the protecting groups, the pentasaccharides were converted into three novel sulfated GM1b gangliosides by the successive introduction of the ceramide and sulfo groups, followed by complete deprotection. Among the synthetic gangliosides, GSC-338 (II3III6-disulfate of iso-GM1b) was surprisingly found to be the most potent MAG binding structure tested to date.
Assuntos
Antígenos de Superfície/química , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/síntese química , Gangliosídeo G(M1)/metabolismo , Gangliosídeos/química , Glicoproteína Associada a Mielina/metabolismo , Animais , Biomimética , Sequência de Carboidratos , Linhagem Celular , Chlorocebus aethiops , Combinação de Medicamentos , Gangliosídeo G(M1)/química , Ligantes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Sulfamonometoxina/química , Trimetoprima/químicaRESUMO
Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids. Analysis has been performed using a novel multivalent platform comprising biotinylated sialosides bound to a streptavidin-alkaline phosphatase conjugate. Each siglec was found to have a unique specificity for binding 16 different sialoside-streptavidin-alkaline phosphatase probes. The relative affinities of monovalent sialosides were assessed for each siglec in competitive inhibition studies. The quantitative data obtained allows a detailed analysis of each siglec for the relative importance of sialic acid and the penultimate oligosaccharide sequence on binding affinity and specificity. Most remarkable was the finding that myelin-associated glycoprotein (Siglec-4) binds with 500-10,000-fold higher affinity to a series of mono- and di-sialylated derivatives of the O-linked T-antigen (Galbeta(1-3)-GalNAc(alpha)OThr) as compared with alpha-methyl-NeuAc.
Assuntos
Moléculas de Adesão Celular , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Imunológicos/metabolismo , Fosfatase Alcalina , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Células CHO , Sequência de Carboidratos , Quimera , Cricetinae , Ensaio de Imunoadsorção Enzimática , Galactosídeos/síntese química , Galactosídeos/metabolismo , Glicolipídeos/síntese química , Glicolipídeos/metabolismo , Glicoproteínas/síntese química , Glicoproteínas/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Receptores Imunológicos/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , EstreptavidinaRESUMO
The interaction of CD22 with glycoprotein ligands bearing the Siaalpha2,6Gal-R sequence is believed to modulate its function as a regulator of B cell signaling. Although a commercial sialoside-polyacrylamide (PAA) probe, NeuAc- alpha2,6Gal-PAA, has facilitated studies on ligand binding by human CD22, murine CD22 binds instead with high affinity to NeuGcalpha2,6Gal-R. A multivalent probe with this sequence was constructed to facilitate investigations of ligand binding in CD22 function using genetically defined murine models. The probe is based on the sialoside-PAA platform, which is then biotinylated for easy detection. A series of sialoside probes were constructed with two different length linker arms between the sialoside and the backbone and three different sialoside to PAA molar ratios. The NeuGcalpha2,6Gal-PAA probe is specific for CD22: it binds to sialidase-treated B cells of wild-type mice but not B cells of CD22-null mice. Additionally, because the probe only binds to sialidase-treated wild-type cells, it confirms that CD22 is constitutively "masked" on most B cells from wild-type mice by binding to ligands in cis. In contrast, the probe bound equally well to native or sialidase-treated B cells from the immunocompromised ligand-deficient ST6Gal I knockout mice, demonstrating that CD22 is constitutively "unmasked" in these cells.