RESUMEN
PURPOSE: Inflammatory bowel disease (IBD) can be imaged with positron emission tomography (PET), but existing PET radiopharmaceuticals have limited diagnostic accuracy. Vascular adhesion protein-1 (VAP-1) is an endothelial cell surface molecule that controls leukocyte extravasation into sites of inflammation. However, the role of inflammation-induced VAP-1 expression in IBD is still unclear. Therefore, this study investigated the utility of VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 positron emission tomography/computed tomography (PET/CT) for assessing inflammation in two mouse models of IBD. PROCEDURES: Studies were performed using K8-/- mice that develop a chronic colitis-phenotype and C57Bl/6NCrl mice with acute intestinal inflammation chemically-induced using 2.5% dextran sodium sulfate (DSS) in drinking water. In both diseased and control mice, uptake of the VAP-1-targeting peptide [68Ga]Ga-DOTA-Siglec-9 was assessed in intestinal regions of interest using in vivo PET/CT, after which ex vivo gamma counting, digital autoradiography, and histopathological analyses were performed. Immunofluorescence staining was performed to determine VAP-1-expression in the intestine, including in samples from patients with ulcerative colitis. RESULTS: Intestinal inflammation could be visualized by [68Ga]Ga-DOTA-Siglec-9 PET/CT in two murine models of IBD. In both models, the in vivo PET/CT and ex vivo studies of [68Ga]Ga-DOTA-Siglec-9 uptake were significantly higher than in control mice. The in vivo uptake was increased on average 1.4-fold in the DSS model and 2.0-fold in the K8-/- model. Immunofluorescence staining revealed strong expression of VAP-1 in the inflamed intestines of both mice and patients. CONCLUSIONS: This study suggests that the VAP-1-targeting [68Ga]Ga-DOTA-Siglec-9 PET tracer is a promising tool for non-invasive imaging of intestinal inflammation. Future studies in patients with IBD and evaluation of the potential value of [68Ga]Ga-DOTA-Siglec-9 in diagnosis and monitoring of the disease are warranted.
Asunto(s)
Compuestos Heterocíclicos con 1 Anillo , Enfermedades Inflamatorias del Intestino , Tomografía Computarizada por Tomografía de Emisión de Positrones , Humanos , Ratones , Animales , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radioisótopos de Galio/química , Modelos Animales de Enfermedad , Tomografía de Emisión de Positrones/métodos , Inflamación , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/farmacologíaRESUMEN
BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',Nâ³,Nâ´-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. METHODS: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. RESULTS: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P = .003) or control rats (SUVmean, 0.2 ± 0.03; P < .0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. CONCLUSION: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions.
Asunto(s)
Miocarditis , Sarcoidosis , Humanos , Ratas , Animales , Porcinos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radioisótopos de Galio/química , Miocarditis/diagnóstico por imagen , Adyuvante de Freund , Tomografía Computarizada por Rayos X , Tomografía de Emisión de Positrones/métodos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/químicaRESUMEN
Environmental pollution, chronic stress, and unhealthy lifestyle are factors that negatively affect reproductive potential. Currently, 15-20% of couples in industrialized countries face the problem of infertility. This growing health and social problem prompts researchers to explore the regulatory mechanisms that may be important for successful fertilization. In recent years, more attention has been paid to male infertility factors, including the impact of seminal plasma components on regulation of the female immune response to allogenic sperm, embryo and fetal antigens. Directing this response to the tolerogenic pathway is crucial to achieve a healthy pregnancy. According to the fetoembryonic defense hypothesis, the regulatory mechanism may be associated with the interaction of lectins and immunomodulatory glycoepitopes. Such interactions may involve lectins of dendritic cells and macrophages, recruited to the cervical region immediately after intercourse. Carbohydrate binding receptors include C type lectins, such as DC-SIGN and MGL, as well as galectins and siglecs among others. In this article we discuss the expression of the possible lectin ligands, highly fucosylated and high mannose structures, which may be recognized by DC-SIGN, glycans of varying degrees of sialylation, which may differ in their interaction with siglecs, as well as T and Tn antigens in O-glycans.
Asunto(s)
Glicoproteínas , Lectinas , Semen , Femenino , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Inmunidad , Lectinas/química , Lectinas/metabolismo , Ligandos , Masculino , Polisacáridos/química , Embarazo , Semen/química , Semen/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismoRESUMEN
Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.
Asunto(s)
Antiinflamatorios/química , Antígenos CD/química , Glicopéptidos/genética , Inflamación/tratamiento farmacológico , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Antiinflamatorios/metabolismo , Antígenos CD/genética , Glicopéptidos/química , Humanos , Macrófagos/efectos de los fármacos , Microglía/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Fagocitos/efectos de los fármacos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Sialic acid-binding immunoglubulinlike lectin 9 (Siglec-9) is a ligand of vascular adhesion protein 1. A 68Ga-labeled peptide of Siglec-9, 68Ga-DOTA-Siglec-9, holds promise as a novel PET tracer for imaging of inflammation. This first-in-humans study investigated the safety, tolerability, biodistribution, and radiation dosimetry of this radiopharmaceutical. Methods: Six healthy men underwent dynamic whole-body PET/CT. Serial venous blood samples were drawn from 1 to 240 min after intravenous injection of 162 ± 4 MBq of 68Ga-DOTA-Siglec-9. In addition to γ-counting, the plasma samples were analyzed by high-performance liquid chromatography to detect intact tracer and radioactive metabolites. Radiation doses were calculated using the OLINDA/EXM software, version 2.2. In addition, a patient with early rheumatoid arthritis was studied with both 68Ga-DOTA-Siglec-9 and 18F-FDG PET/CT to determine the ability of the new tracer to detect arthritis. Results:68Ga-DOTA-Siglec-9 was well tolerated by all subjects. 68Ga-DOTA-Siglec-9 was rapidly cleared from the blood circulation, and several radioactive metabolites were detected. The organs with the highest absorbed doses were the urinary bladder wall (0.38 mSv/MBq) and kidneys (0.054 mSv/MBq). The mean effective dose was 0.022 mSv/MBq (range, 0.020-0.024 mSv/MBq). Most importantly, however, 68Ga-DOTA-Siglec-9 was comparable to 18F-FDG in detecting arthritis. Conclusion: Intravenous injection of 68Ga-DOTA-Siglec-9 was safe and biodistribution was favorable for testing of the tracer in larger group of patients with rheumatoid arthritis, as is planned for the next phase of clinical trials. The effective radiation dose of 68Ga-DOTA-Siglec-9 was within the same range as the effective radiation doses of other 68Ga-labeled tracers. Injection of 150 MBq of 68Ga-DOTA-Siglec-9 would expose a subject to 3.3 mSv. These findings support the possible repeated clinical use of 68Ga-DOTA-Siglec-9, such as in trials to elucidate the treatment efficacy of novel drug candidates.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Antígenos CD/química , Moléculas de Adhesión Celular/metabolismo , Radioisótopos de Galio/química , Compuestos Heterocíclicos con 1 Anillo/química , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiofármacos/química , Radiofármacos/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Adulto , Femenino , Humanos , Ligandos , Masculino , Radiofármacos/efectos adversos , Radiofármacos/farmacocinética , Seguridad , Solubilidad , Distribución TisularRESUMEN
The sialic acid-binding immunoglobulin-type of lectins (Siglecs) are receptors that recognize sialic acid-containing glycans. In the majority of the cases, Siglecs are expressed on immune cells and play a critical role in regulating immune cell signaling. Over the years, it has been shown that the sialic acid-Siglec axis participates in immunological homeostasis, and that any imbalance can trigger different pathologies, such as autoimmune diseases or cancer. For all this, different therapeutics have been developed that bind to Siglecs, either based on antibodies or being smaller molecules. In this review, we briefly introduce the Siglec family and we compile a description of glycan-based molecules and antibody-based therapies (including CAR-T and bispecific antibodies) that have been designed to therapeutically targeting Siglecs.
Asunto(s)
Terapia Molecular Dirigida , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Animales , Anticuerpos/metabolismo , Humanos , Nanopartículas/química , Polisacáridos/química , Polisacáridos/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/químicaRESUMEN
Alzheimer's disease (AD) is characterized by the appearance of amyloid-ß plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV-2 cell line and human stem cell-derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. Collectively, our results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration.
Asunto(s)
Inflamación/patología , Microglía/metabolismo , Degeneración Nerviosa/patología , Fosfoproteínas/metabolismo , Proteómica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Muerte Celular , Línea Celular , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Ratones Transgénicos , Microglía/patología , Degeneración Nerviosa/metabolismo , Péptidos/metabolismo , Fagocitosis , Fosfotirosina/metabolismo , Proteoma/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Transducción de Señal , Regulación hacia ArribaRESUMEN
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer's disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.
Asunto(s)
Polisacáridos/análisis , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias de la Mama/metabolismo , Células CHO , Cricetulus , Femenino , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Células K562 , Espectrometría de Masas , Polisacáridos/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/aislamiento & purificación , Ácidos Siálicos/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Bazo/citología , Bazo/metabolismo , Estreptavidina/metabolismoRESUMEN
Sialic acid-binding immunoglobulin-like lectins (Siglec)-like domains of streptococcal serine-rich repeat (SRR) adhesins recognize sialylated glycans on human salivary, platelet, and plasma glycoproteins via a YTRY sequence motif. The SRR adhesin from Streptococcus sanguinis strain SK1 has tandem sialoglycan-binding domains and has previously been shown to bind sialoglycans with high affinity. However, both domains contain substitutions within the canonical YTRY motif, making it unclear how they interact with host receptors. To identify how the S. sanguinis strain SK1 SRR adhesin affects interactions with sialylated glycans and glycoproteins, we determined high-resolution crystal structures of the binding domains alone and with purified trisaccharides. These structural studies determined that the ligands still bind at the noncanonical binding motif, but with fewer hydrogen-bonding interactions to the protein than is observed in structures of other Siglec-like adhesins. Complementary biochemical studies identified that each of the two binding domains has a different selectivity profile. Interestingly, the binding of SK1 to platelets and plasma glycoproteins identified that the interaction to some host targets is dominated by the contribution of one binding domain, whereas the binding to other host receptors is mediated by both binding domains. These results provide insight into outstanding questions concerning the roles of tandem domains in targeting host receptors and suggest mechanisms for how pathogens can adapt to the availability of a range of related but nonidentical host receptors. They further suggest that the definition of the YTRY motif should be changed to ÏTRX, a more rigorous description of this sialic acid-recognition motif given recent findings.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Glicoproteínas/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Infecciones Estreptocócicas/metabolismo , Streptococcus sanguis/fisiología , Adhesinas Bacterianas/química , Secuencias de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Glicoproteínas/química , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Conformación Proteica , Dominios Proteicos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Streptococcus sanguis/químicaRESUMEN
Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
Asunto(s)
Inmunoterapia/métodos , Melanoma Experimental/terapia , Neuraminidasa/inmunología , Polisacáridos/química , Receptor ErbB-2/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Aloinjertos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Humanos , Hidrólisis , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Inmunoconjugados/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Terapia Molecular Dirigida , Neuraminidasa/química , Neuraminidasa/genética , Polisacáridos/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Análisis de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The cellular glycocalyx of vertebrates is frequently decorated with sialic acid residues. These sialylated structures are recognized by sialic acid-binding immunoglobulin-type lectins (Siglecs) of immune cells, which modulate their responsiveness. Fifteen Siglecs are known to be expressed in humans, but only four Siglecs are regularly present in fish: Siglec1, CD22, myelin-associated glycoprotein (MAG), and Siglec15. While several studies have dealt with the physiological roles of these four Siglecs in mammals, little is known about Siglecs in fish. In the present manuscript, the expression landscapes of these Siglecs were determined in the two salmonid species Oncorhynchus mykiss and Coregonus maraena and in the percid fish Sander lucioperca. This gene-expression profiling revealed that the expression of MAG is not restricted to neuronal cells but is detectable in all analyzed blood cells, including erythrocytes. The teleostean MAG contains the inhibitory motif ITIM; therefore, an additional immunomodulatory function of MAG is likely to be present in fish. Besides MAG, Siglec1, CD22, and Siglec15 were also expressed in all analyzed blood cell populations. Interestingly, the expression profiles of genes encoding Siglecs and particular associated enzymes changed in a gene- and tissue-specific manner when Coregonus maraena was exposed to handling stress. Thus, the obtained data indicate once more that stress directly affects immune-associated processes.
Asunto(s)
Peces/genética , Regulación de la Expresión Génica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Secuencia de Aminoácidos , Animales , Dosificación de Gen , Perfilación de la Expresión Génica , Humanos , Ratones , Modelos Moleculares , Especificidad de Órganos/genética , Homología de Secuencia de Aminoácido , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Especificidad de la Especie , Estrés Psicológico/genéticaRESUMEN
The function of natural killer (NK) cells, defending against virus infection and tumour progression, is regulated by multiple activating and inhibiting receptors expressed on NK cells, among which sialic acid-bind immunoglobulin-like lectins (Siglecs) act as a vital inhibitory group. Previous studies have shown that Siglec7 and Siglec9 are expressed on NK cells, which negatively regulate the function of NK cells and modulate the immune response through the interaction of sialic acid-containing ligands. Siglec7 and Siglec9 are very similar in distribution, gene encoding, protein sequences, ligand affinity, and functions in regulating the immune system against virus and cancers, but differences still exist between them. In this review, we aim to discuss the similarities and differences between Siglec7 and Siglec9 and analyze their functions in virus infection and tumour progression in order to develop better anti-viral and anti-tumor immunotherapy in the future.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Células Asesinas Naturales/inmunología , Lectinas/fisiología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/fisiología , Virosis/inmunología , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/química , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Progresión de la Enfermedad , Humanos , Células Asesinas Naturales/metabolismo , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Virosis/metabolismo , Virosis/patologíaRESUMEN
Siglecs are transmembrane receptor-like vertebrate lectins that recognize glycans containing sialic acid. Most Siglecs also interact with intracellular signal transduction molecules, and modulate immune responses. Recombinant soluble Siglecs fused with the fragment crystallizable (Fc) region of immunoglobulin G (Siglec-Fc) are a versatile tool for the investigation of Siglec functions. We describe protocols for the production of recombinant Siglec-Fc, the analysis of expression of Siglec ligands by flow cytometry, and the identification of the Siglec ligand candidates based on proximity labeling.
Asunto(s)
Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ácidos Siálicos/metabolismo , Biotina/química , Citometría de Flujo , Humanos , Ligandos , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Transducción de Señal , Coloración y EtiquetadoRESUMEN
In order to combat invading pathogens neutrophils can release neutrophil extracellular traps (NETs). However, since NETs can also damage endogenous cells, several control mechanisms for the formation of NETs must work effectively. For instance, neutrophil activation is silenced within blood circulation by the binding of sialylated glycoconjugates to sialic acid binding immunoglobulin-like lectins (Siglecs) on neutrophils. As neutrophils are recruited within the female reproductive tract, after mating, a comparable mechanism may also take place within the bovine cervix to prevent an exaggerated NET formation and thus, infertility. We examined, if the highly glycosylated mucins, which are the major functional fraction of biomolecules in mucus, represent a potential regulator of NET formation. The qPCR data revealed that in polymorphonuclear neutrophils (PMNs) inhibitory Siglecs are the most frequently expressed Siglecs and might be a potential target of sialylated glycans to modulate the activation of PMNs. Remarkably, the addition of bovine cervical mucins significantly inhibited the formation of NET, which had been induced in response to lipopolysaccharides (LPS) or a combination of phorbol myristate acetate (PMA) and ionomycin. The inhibitory effects were independent of the stage of estrous cycle (estrus, luteal, and follicular mucins). PMNs retained their segmented nuclei and membrane perforation was prevented. However, the inhibitory effects were diminished, when sialic acids were released under acidic conditions. Comparable results were achieved, when sialic acids were targeted by neuraminidase digestion, indicating a sialic acid dependent inhibition of NET release. Thus, bovine cervical mucins have an anti-inflammatory capability to modulate NET formation and might be further immunomodulatory biomolecules that support fertility.
Asunto(s)
Cuello del Útero/metabolismo , Trampas Extracelulares/metabolismo , Mucinas/metabolismo , Activación Neutrófila , Neutrófilos/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cuello del Útero/inmunología , Trampas Extracelulares/inmunología , Femenino , Expresión Génica , Glicosilación , Hidrólisis , Inmunohistoquímica , Modelos Moleculares , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Relación Estructura-ActividadRESUMEN
Siglecs are mammalian sialic acid (Sia) recognizing immuno-globulin-like receptors expressed across the major leukocyte lineages, and function to recognize ubiquitous Sia epitopes on the cell surface. Many Siglecs are inhibitory receptors expressed on innate immune cells, they also have a role in maintaining B cell tolerance as well as modulating the activation of conventional and plasmocytic dendritic cells. Through these and other roles they contribute directly and indirectly to the regulation of T cell function. Siglecs have been identified to play key roles in several forms of blood cancers, autoimmune and infection deceases. So far as we know, there's no Siglecs related research works on solid organ transplantation. In this review, we describe our understanding of the potential roles of Siglecs in the regulation of immune cell function, which may be crosslinked to allo-rejection and ischemia-reperfusion injury.
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Sistema Inmunológico/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Animales , Autoinmunidad , Linfocitos B/inmunología , Células Dendríticas/inmunología , Rechazo de Injerto/inmunología , Humanos , Sistema Inmunológico/patología , Inmunidad Innata , Células Asesinas Naturales/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Linfocitos T/inmunologíaRESUMEN
Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.
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Antígenos CD/química , Antígenos de Diferenciación Mielomonocítica/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Lectinas/química , Metaloendopeptidasas/química , Mucinas/química , Proteínas de Neoplasias/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Secuencias de Aminoácidos , Humanos , Espectrometría de Masas , Especificidad por SustratoRESUMEN
Although a plethora of players has already been revealed to be engaged in the haemostatic system, a fundamental consideration of the molecular nature of information coding can give further explorations of the mechanisms of blood clotting, platelet functionality and vascular trafficking direction. By any measures, looking at ranges of occurrence and of potential for structural versatility, at strategic positioning to influence protein and cell sociology as well as at dynamics of processing and restructuring for phenotypic variability, using sugars as an alphabet of life for generating the glycan part of glycoconjugates is a success story. The handiwork by the complex system for glycan biosynthesis renders biochemical messages of exceptionally high coding capacity available. They are read and translated into cellular effects by receptors termed lectins. The different levels of regulation on both sides, that is, glycan and lectin, establish an intriguingly fine-tuned capacity for functional pairing. The emerging insights into the highly branched routes of glycosylation, into lectin structures up to complete characterization in solution and the shape of lectin networks, first obtained for the three selectins, now extended to considering many other C-type lectins, galectins and siglecs, as well as into intra- and inter-family cross-talk and cooperations are sure to push boundaries in our understanding of the molecular basis of haemostasis.
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Vasos Sanguíneos/fisiología , Lectinas/química , Polisacáridos/química , Transducción de Señal , Empalme Alternativo , Animales , Carbohidratos/química , Sistema Cardiovascular , Membrana Celular/química , Epítopos/química , Galectinas/química , Glicosilación , Hemostasis , Humanos , Inflamación , Conformación Molecular , Fenotipo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/químicaRESUMEN
Streptococcus gordonii and Streptococcus sanguinis are typically found among the normal oral microbiota but can also cause infective endocarditis. These organisms express cell surface serine-rich repeat adhesins containing "Siglec-like" binding regions (SLBRs) that mediate attachment to α2-3-linked sialic acids on human glycoproteins. Two known receptors for the Siglec-like adhesins are the salivary mucin MG2/MUC7 and platelet GPIbα, and the interaction of streptococci with these targets may contribute to oral colonization and endocarditis, respectively. The SLBRs display a surprising diversity of preferences for defined glycans, ranging from highly selective to broader specificity. In this report, we characterize the glycoproteins in human plasma recognized by four SLBRs that prefer different α2-3 sialoglycan structures. We found that the SLBRs recognize a surprisingly small subset of plasma proteins that are extensively O-glycosylated. The preferred plasma protein ligands for a sialyl-T antigen-selective SLBR are proteoglycan 4 (lubricin) and inter-alpha-trypsin inhibitor heavy chain H4. Conversely, the preferred ligand for a 3'sialyllactosamine-selective SLBR is glycocalicin (the extracellular portion of platelet GPIbα). All four SLBRs recognize C1 inhibitor but detect distinctly different glycoforms of this key regulator of the complement and kallikrein protease cascades. The four plasma ligands have potential roles in thrombosis and inflammation, and each has been cited as a biomarker for one or more vascular or other diseases. The combined results suggest that the interaction of Siglec-like adhesins with different subsets of plasma glycoproteins could have a significant impact on the propensity of streptococci to establish endocardial infections.
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Proteínas Bacterianas/química , Proteínas Sanguíneas/química , Endocarditis , Glicoproteínas/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Streptococcus gordonii/química , Streptococcus sanguis/química , Proteínas Bacterianas/metabolismo , Proteínas Sanguíneas/metabolismo , Glicoproteínas/metabolismo , Humanos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Streptococcus gordonii/metabolismo , Streptococcus sanguis/metabolismoRESUMEN
Molecular signals on the cell surface are responsible for adhesion and communication. Of relevance in this respect, their chemical properties endow carbohydrates with the capacity to store a maximum of information in a minimum of space. One way to present glycans on the cell surface is their covalent conjugation to a ceramide anchor. Among the resulting glycosphingolipids, gangliosides are special due to the presence of at least one sialic acid in the glycan chains. Their spatial accessibility and the dynamic regulation of their profile are factors that argue in favor of a role of glycans of gangliosides as ligands (counterreceptors) for carbohydrate-binding proteins (lectins). Indeed, as discovered first for a bacterial toxin, tissue lectins bind gangliosides and mediate contact formation (trans) and signaling (cis). While siglecs have a preference for higher sialylated glycans, certain galectins also target the monosialylated pentasaccharide of ganglioside GM1. Enzymatic interconversion of ganglioside glycans by sialidase action, relevant for neuroblastoma cell differentiation and growth control in vitro, for axonogenesis and axon regeneration, as well as for proper communication between effector and regulatory T cells, changes lectin-binding affinity profoundly. The GD1a-to-GM1 "editing" is recognized by such lectins, for example, myelin-associated glycoprotein (siglec-4) losing affinity and galectin-1 gaining reactivity, and then translated into postbinding signaling. Orchestrations of loss/gain of affinity, of ganglioside/lectin expression, and of lectin presence in a network offer ample opportunities for fine-tuning. Thus glycans of gangliosides such as GD1a and GM1 are functional counterreceptors by a pairing with tissue lectins, an emerging aspect of ganglioside and lectin functionality.
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Galectinas/metabolismo , Gangliósidos/metabolismo , Enfermedades Metabólicas/fisiopatología , Polisacáridos/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Animales , Galectinas/química , Gangliósidos/química , Humanos , Ligandos , Polisacáridos/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Transducción de SeñalRESUMEN
Human primary amine oxidase (hAOC3), also known as vascular adhesion protein 1, mediates leukocyte rolling and trafficking to sites of inflammation by a multistep adhesion cascade. hAOC3 is absent on the endothelium of normal tissues and is kept upregulated during inflammatory conditions, which is an applicable advantage for imaging inflammatory diseases. Sialic acid binding immunoglobulin like-lectin 9 (Siglec-9) is a leukocyte ligand for hAOC3. The peptide (CARLSLSWRGLTLCPSK) based on the region of Siglec-9 that interacts with hAOC3, can be used as a specific tracer for hAOC3-targeted imaging of inflammation using Positron Emission Tomography (PET). In the present study, we show that the Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase activity towards benzylamine. Furthermore, the hAOC3 inhibitors semicarbazide and imidazole reduce the binding of wild type and Arg/Ala mutated Siglec-9 peptides to hAOC3. Molecular docking of the Siglec-9 peptide is in accordance with the experimental results and predicts that the R3 residue in the peptide interacts in the catalytic site of hAOC3 when the topaquinone cofactor is in the non-catalytic on-copper conformation. The predicted binding mode of Siglec-9 peptide to hAOC3 is supported by the PET studies using rodent, rabbit and pig AOC3 proteins.