RESUMEN
N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.
Asunto(s)
Glicoproteínas , Sialiltransferasas , Animales , Técnicas de Inactivación de Genes , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/farmacocinética , Glicosilación , Células HEK293 , Humanos , Ratas , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indicating that rFVIII-Fc is capable of amplifying the maturation signal. On the contrary, cultivation of moDC's with rFVIII did not alter cell morphology or increase surface activation marker expression and pro-inflammatory cytokine production. The binding of the Fc domain to the activating Fcγ receptor IIa (FcγRIIa) can cause cell activation. Therefore, the effect of rFVIII-Fc on FcγRIIa was analyzed in detail. Cultivation of moDC's with rFVIII-Fc led to increased phosphorylation of FcγRIIa, which was not detected for rFVIII. Blocking FcγRIIa prior to the cultivation with rFVIII-Fc significantly reduced the activating effect of rFVIII-Fc, indicating that rFVIII-Fc-induced moDC activation was caused by FcγRIIa. Moreover, rFVIII-Fc bound to FCGR2A-transfected human embryonic kidney 293 cells. Taken together, our data present a new mechanism of moDC activation by rFVIII-Fc via FcγRIIa.
RESUMEN
Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor's quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor's properties, most notably its binding to Fcγ receptors.
RESUMEN
Panzyga® is a new glycine-formulated immune globulin intravenous 10% liquid for the treatment of patients suffering from immunodeficiencies and autoimmune diseases. Panzyga® is a high purity, native and functional IgG product with an IgG subclass distribution equivalent to normal plasma. The levels of hemagglutinins and accompanying plasma proteins (including IgA and IgM) are low. Potential procoagulant activity is not detectable. Functional activity of the IgG was demonstrated by opsonophagocytosis and receptor binding assays. Dynamic light scattering measurements and fluorescent dye binding were used to characterize the integrity of the IgG molecule. Panzyga® is stable under refrigerated storage for at least two years regarding all assessed physicochemical and functional parameters; it can also be stored at room temperature for at least twelve months within its total shelf-life.
Asunto(s)
Frío , Almacenaje de Medicamentos/métodos , Inmunoglobulina G/química , Inmunoglobulinas Intravenosas/química , Adulto , Cromatografía Líquida de Alta Presión , Ensayos Clínicos Fase III como Asunto , Estabilidad de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulina G/uso terapéutico , Inmunoglobulinas Intravenosas/metabolismo , Inmunoglobulinas Intravenosas/uso terapéutico , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Oxazinas/metabolismo , Unión Proteica , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Sialuria is a rare autosomal dominant disorder of mammalian metabolism, caused by defective feedback inhibition of the UDP-N-acetylglucosamine-2-epimerase N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. Sialuria is characterized by overproduction of free sialic acid in the cell cytoplasm. Patients exhibit vastly increased urinary excretion of sialic acid and show differently pronounced developmental delays. The physiopathology of sialuria is not well understood. Here we established a transgenic mouse line that expresses GNE containing the sialuria mutation R263L, in order to investigate the influence of an altered sialic acid concentration on the organism. The transgenic mice that expressed the mutated RNA excreted up to 400 times more N-acetylneuraminic acid than wild-type mice. Additionally, we found higher sialic acid concentration in the brain cytoplasm. Analyzing the (poly)sialylation of neural cell adhesion molecule (NCAM) revealed increased polysialylation in brains of transgenic mice compared to wild-type. However, we found only minor changes in membrane-bound sialylation in various organs but, surprisingly, a significant increase in surface sialylation on leukocytes. Our results suggest that the intracellular sialic acid concentration regulates polysialylation on NCAM in vivo; this could play a role in the manifestation of the developmental delays in sialuria patients.
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Leucocitos/metabolismo , Complejos Multienzimáticos/genética , Ácido N-Acetilneuramínico/orina , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Procesamiento Proteico-Postraduccional , Enfermedad por Almacenamiento de Ácido Siálico/metabolismo , Factores de Edad , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Humanos , Leucocitos/patología , Hígado/metabolismo , Ratones , Ratones Transgénicos , Complejos Multienzimáticos/deficiencia , Mutación , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Especificidad de Órganos , Enfermedad por Almacenamiento de Ácido Siálico/genética , Enfermedad por Almacenamiento de Ácido Siálico/patologíaAsunto(s)
Factor VIII/genética , Factor VIII/uso terapéutico , Hemofilia A/terapia , Animales , HumanosRESUMEN
Due to its position at the outermost of glycans, sialic acid is involved in a myriad of physiological and pathophysiological cell functions such as host-pathogen interactions, immune regulation, and tumor evasion. Inhibitors of cell surface sialylation could be a useful tool in cancer, immune, antibiotic, or antiviral therapy. In this work, four different C-3 modified N-acetylmannosamine analogs were tested as potential inhibitors of cell surface sialylation. Peracetylated 2-acetylamino-2-deoxy-3-O-methyl-D-mannose decreases cell surface sialylation in Jurkat cells in a dose-dependent manner up to 80%, quantified by flow cytometry and enzyme-linked lectin assays. High-performance liquid chromatography experiments revealed that not only the concentration of membrane bound but also of cytosolic sialic acid is reduced in treated cells. We have strong evidence that the observed reduction of sialic acid expression in cells is caused by the inhibition of the bifunctional enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase. 2-Acetylamino-2-deoxy-3-O-methyl-D-mannose inhibits the human ManNAc kinase domain of the UDP-GlcNAc-2-epimerase/ManNAc kinase. Binding kinetics of the inhibitor and human N-acetylmannosamine kinase were evaluated using surface plasmon resonance. Specificity studies with human N-acetylglucosamine kinase and hexokinase IV indicated a high specificity of 2-acetylamino-2-deoxy-3-O-methyl-D-mannose for MNK. This substance represents a novel class of inhibitors of sialic acid expression in cells, targeting the key enzyme of sialic acid de novo biosynthesis.
Asunto(s)
Carbohidrato Epimerasas/química , Proteínas Portadoras/química , Hexosaminas/química , Ácido N-Acetilneuramínico/química , Cromatografía Líquida de Alta Presión , Clonación Molecular , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Células Jurkat , Cinética , Lectinas , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Unión Proteica , Dispersión de Radiación , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: Sialic acids (Sia) represent negative-charged terminal sugars on most glycoproteins and glycolipids on the cell surface of vertebrates. Aberrant expression of tumor associated sialylated carbohydrate epitopes significantly increases during onset of cancer. Since Sia contribute towards cell migration (â=â metastasis) and to chemo- and radiation resistance. Modulation of cellular Sia concentration and composition poses a challenge especially for neuroblastoma therapy, due to the high heterogeneity and therapeutic resistance of these cells. Here we propose that Metabolic Sia Engineering (MSE) is an effective strategy to reduce neuroblastoma progression and metastasis. METHODS: Human neuroblastoma SH-SY5Y cells were treated with synthetic Sia precursors N-propanoyl mannosamine (ManNProp) or N-pentanoyl mannosamine (ManNPent). Total and Polysialic acids (PolySia) were investigated by high performance liquid chromatography. Cell surface polySia were examined by flow-cytometry. Sia precursors treated cells were examined for the migration, invasion and sensitivity towards anticancer drugs and radiation treatment. RESULTS: Treatment of SH-SY5Y cells with ManNProp or ManNPent (referred as MSE) reduced their cell surface sialylation significantly. We found complete absence of polysialylation after treatment of SH-SY5Y cells with ManNPent. Loss of polysialylation results in a reduction of migration and invasion ability of these cells. Furthermore, radiation of Sia-engineered cells completely abolished their migration. In addition, MSE increases the cytotoxicity of anti-cancer drugs, such as 5-fluorouracil or cisplatin. CONCLUSIONS: Metabolic Sia Engineering (MSE) of neuroblastoma cells using modified Sia precursors reduces their sialylation, metastatic potential and increases their sensitivity towards radiation or chemotherapeutics. Therefore, MSE may serve as an effective method to treat neuroblastoma.
Asunto(s)
Ingeniería Metabólica , Ácido N-Acetilneuramínico/metabolismo , Neuroblastoma/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Citometría de Flujo , Hexosaminas/metabolismo , Hexosaminas/farmacología , Humanos , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacología , Neuroblastoma/tratamiento farmacológico , Tolerancia a Radiación/efectos de los fármacos , Ácidos Siálicos/metabolismo , Ácidos Siálicos/farmacologíaRESUMEN
INTRODUCTION: Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galß1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans. MATERIALS AND METHODS: We describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection. RESULTS AND CONCLUSIONS: Human-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or α-Gal epitopes observed in Chinese Hamster Ovary- and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products.
Asunto(s)
Factor VIII/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Antígenos , Células CHO , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Cricetinae , Cricetulus , Factor VIII/química , Factor VIII/genética , Factor VIII/inmunología , Glicosilación , Células HEK293 , Humanos , Espectrometría de Masas , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/metabolismo , Sulfatos/metabolismo , Transfección , TirosinaRESUMEN
Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac(2)) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac(2)). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac(2) was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac(2)9Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac(2), relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac(2) was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.
Asunto(s)
Coronavirus/metabolismo , Hemaglutininas Virales/metabolismo , Ácido N-Acetilneuramínico/análisis , Ácido N-Acetilneuramínico/metabolismo , Proteínas Virales de Fusión/metabolismo , Acetilación , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Coronavirus/patogenicidad , Femenino , Cromatografía de Gases y Espectrometría de Masas/métodos , Masculino , Ratones , Ratones Endogámicos , Ácido N-Acetilneuramínico/química , Especificidad por SustratoRESUMEN
Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.
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Leucocitos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Sialoglicoproteínas/química , Sialoglicoproteínas/aislamiento & purificación , Adolescente , Niño , Preescolar , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Valor Predictivo de las Pruebas , Pronóstico , Ácidos Siálicos/síntesis química , Ácidos Siálicos/farmacología , Sialoglicoproteínas/antagonistas & inhibidoresRESUMEN
The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate-7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotide-sugar level [Shen et al., Biol. Chem. 383 (2002), 307-317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMP-Neu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-N-acetyl-9-O-acetyl-neuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2 could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMP-Neu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galbeta1,3(4)R- residues.
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Colon/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Mucosa Intestinal/metabolismo , Acetilcoenzima A/antagonistas & inhibidores , Acetilcoenzima A/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Animales , Asialoglicoproteínas/química , Asialoglicoproteínas/metabolismo , Bovinos , Colon/química , Ácido N-Acetilneuramínico Citidina Monofosfato/análogos & derivados , Ácido N-Acetilneuramínico Citidina Monofosfato/antagonistas & inhibidores , Fetuínas , Glicoconjugados/química , Glicoconjugados/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Glicoproteínas de Membrana/metabolismo , Mucinas/química , Mucinas/metabolismo , Transporte de Proteínas , Ratas , Sialiltransferasas/metabolismo , Glándula Submandibular/química , Vesículas Transportadoras/metabolismo , alfa-Fetoproteínas/metabolismo , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
A decrease in the level of O-acetylated sialic acids observed in colorectal carcinoma may lead to an increase in the expression of sialyl Lewis(X), a tumor-associated antigen, which is related to progression of colorectal cancer to metastasis. The underlying mechanism for this reduction is, however, not fully understood. Two enzymes are thought to be primarily responsible for the turnover of O-acetyl ester groups on sialic acids; sialate-O-acetyltransferase (OAT) and sialate-O-acetylesterase (OAE). We have previously reported the characterization of OAT activity from normal colon mucosa, which efficiently O-acetylates CMP-Neu5Ac exclusively in the Golgi apparatus prior to the action of sialyltransferase. In this report we describe the identification of a lysosomal and a cytosolic OAE activity in human colonic mucosa that specifically hydrolyses 9-O-acetyl groups on sialic acid. Utilizing matched resection margin and cancer tissue from colorectal carcinoma patients we provide strong evidence suggesting that the level of O-acetylated sialic acids present in normal and diseased human colon may be dependent on the relative activities of OAT to lysosomal OAE. Furthermore, we show that the level of free cytosolic Neu5,9Ac2 in human colon is regulated by the relative activity of the cytosolic OAE.
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Acetiltransferasas/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Neoplasias Colorrectales/enzimología , Ácido N-Acetilneuramínico/metabolismo , Ácidos Siálicos/metabolismo , Acetilación , Acetilesterasa , Estudios de Casos y Controles , Cromatografía Líquida de Alta Presión , Colon/enzimología , Citosol/enzimología , Cromatografía de Gases y Espectrometría de Masas , Humanos , Lisosomas/enzimologíaRESUMEN
The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate-4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme O-acetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble O-acetyltransferase maximally operated at 30 degrees C, pH 5.6, and 50-70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 microM for AcCoA and 1.2 microM for GD3. CoA inhibits the enzyme with a Ki value of 14.8 microM. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration.
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Acetiltransferasas/metabolismo , Hígado/enzimología , Acetilcoenzima A/metabolismo , Acetilación , Acetiltransferasas/química , Animales , Ácidos Cólicos , Cromatografía en Capa Delgada , Gangliósidos/metabolismo , Aparato de Golgi/enzimología , Cobayas , Cinética , Sialiltransferasas/metabolismo , Solubilidad , Especificidad por Sustrato , UltrafiltraciónRESUMEN
In the present study a primary culture system of chicken embryo brain neurons was used in the early period of chicken brain development from day 6 until day 8, which was shown to be a suitable model of neuritogenesis, cell migration and reaggregation. Dissociated chicken optic tectum cells from embryonic stage 31 were cultured on polylysine-coated dishes under serum-free conditions up to 3 days. Freshly dissociated neurons developed short processes, which contacted one another and formed fasciculated bundles. Cell somata migrated along the neurite bundles, similar to migrating neurons in vivo, forming three-dimensional tissue-like clusters. This system was used to study the possible functions of the disialoganglioside GD3 for these neuronal differentiation steps. GD3 represents the predominant ganglioside of embryonic neurons before neuritogenesis in vitro and in vivo. Its biosynthesis is followed during day 6 until day 8 of embryonic brain development. Incubation of dissociated neurons with the monoclonal antibody R-24, recognising the GD3 on the cell surface, led to a total blocking of neurite outgrowth. Accordingly, neither cell migration nor reaggregation could be found. These results indicate that the disialoganglioside GD3 plays a central role in neuronal differentiation and development in the embryonic chicken brain.
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Encéfalo/metabolismo , Gangliósidos/metabolismo , Animales , Anticuerpos Monoclonales , Encéfalo/citología , Encéfalo/embriología , Secuencia de Carbohidratos , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Embrión de Pollo , Gangliósidos/química , Gangliósidos/inmunología , Datos de Secuencia Molecular , Neuritas/ultraestructura , Neuronas/citología , Neuronas/metabolismoRESUMEN
Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.
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Glicoconjugados/análisis , Leishmania donovani/fisiología , Ácidos Siálicos/análisis , Acetilesterasa , Pruebas de Aglutinación , Animales , Sangre , Western Blotting , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Cromatografía de Gases y Espectrometría de Masas , Interacciones Huésped-Parásitos , Lectinas , Leishmania donovani/química , Neuraminidasa , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues.
Asunto(s)
Gangliósidos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Tumores Neuroectodérmicos/metabolismo , Acetilación , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Humanos , Datos de Secuencia Molecular , Neuronas/metabolismoRESUMEN
Inflammatory processes often lead to pathologic changes in the area of the larynx. A moistening function of the false vocal folds has been described frequently. Up to now we have little knowledge of the role of the false vocal folds in protection against pathogenic agents. The present study analyzes the structures of the false vocal folds in their relations to antimicrobial defense mechanisms. Investigations were performed on false vocal folds of larynges from 34 cadavers using histologic, histochemical and immunohistochemical methods. Seromucous glands, together with epithelial and goblet cells of the folds, synthesize a complex mucus layer. In all of the investigated samples this layer contains carbohydrates including N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, mannose, fucose, and sialic acids. Furthermore, antimicrobial peptides like lactoferrin, lysozyme, alpha and beta defensins are also found in these structures. IgA, produced by plasma cells in the false vocal folds, is frequently integrated in the secretory product. Synthesized mucins, antimicrobial peptides and immunoglobulins form a specialized protective substance that is secreted mainly at the true vocal folds. Here the layer functions to lubricate the true vocal folds, resulting in positive functional consequences during vocal production. Moreover, together with immunocompetent cells, the protective layer seems to play a major role in antigen defense and prevents invasion of pathogenic agents.