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1.
Biol Pharm Bull ; 47(1): 334-338, 2024 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-38143078

RESUMEN

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.


Asunto(s)
Inmunoglobulina G , Receptores de IgG , Receptores de IgG/química , Receptores de IgG/metabolismo , Rituximab/farmacología , Microscopía de Fuerza Atómica , Unión Proteica , Factores Inmunológicos , Proteínas Portadoras/metabolismo
2.
Biotechnol Lett ; 39(9): 1299-1308, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28547344

RESUMEN

OBJECTIVE: To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. RESULTS: Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. CONCLUSIONS: Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.


Asunto(s)
Acetilglucosaminidasa/biosíntesis , Bombyx/enzimología , Bombyx/metabolismo , Silenciador del Gen , Proteínas Recombinantes/metabolismo , Acetilglucosaminidasa/genética , Animales , Bombyx/genética , Expresión Génica , Vectores Genéticos , Glicosilación , Nucleopoliedrovirus/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética
3.
Glycoconj J ; 33(3): 405-415, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26511985

RESUMEN

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.


Asunto(s)
Glicómica/métodos , Espectrometría de Masas/métodos , Técnicas de Diagnóstico Molecular/métodos , Polisacáridos/química , Biomarcadores/química , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Glicómica/normas , Glicoproteínas/química , Humanos , Espectrometría de Masas/normas , Técnicas de Diagnóstico Molecular/normas , Proteómica/métodos , Proteómica/normas , Reproducibilidad de los Resultados
4.
J Biomol NMR ; 62(2): 157-67, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25902760

RESUMEN

Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing (15)N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a (15)N-enrichment ratio of approximately 80%. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.


Asunto(s)
Bombyx/genética , Glicoproteínas/química , Inmunoglobulina G/química , Marcaje Isotópico/métodos , Animales , Baculoviridae , Cromatografía Liquida , Regulación de la Expresión Génica , Glicoproteínas/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Larva , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masas en Tándem
5.
Plant Cell Rep ; 34(6): 959-68, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25689888

RESUMEN

KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.


Asunto(s)
Nicotiana/genética , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Adalimumab/genética , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Glicosilación , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Nicotiana/metabolismo
6.
Glycobiology ; 24(2): 125-38, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24100142

RESUMEN

After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N-glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N-glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.


Asunto(s)
Islotes Pancreáticos/metabolismo , Polisacáridos/química , Porcinos , Animales , Animales Modificados Genéticamente , Secuencia de Carbohidratos , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Técnicas de Inactivación de Genes , Humanos , Datos de Secuencia Molecular , Polisacáridos/metabolismo , Porcinos/genética , Porcinos/metabolismo
7.
J Biol Chem ; 285(27): 20793-805, 2010 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-20439988

RESUMEN

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO(4))) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO(4)) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO(4)) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO(4)) residues in both CS and DS. IdoA-GalNAc(4,6-SO(4)) units that account for approximately 40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO(4)) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO(4)) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.


Asunto(s)
Acetilgalactosamina/análogos & derivados , Médula Ósea/enzimología , Sulfatos de Condroitina/biosíntesis , Dermatán Sulfato/biosíntesis , Glicosaminoglicanos/biosíntesis , Péptido Hidrolasas/metabolismo , ARN Mensajero/genética , Sulfotransferasas/deficiencia , Acetilgalactosamina/biosíntesis , Acetilgalactosamina/química , Animales , Médula Ósea/ultraestructura , Sulfatos de Condroitina/química , ADN/genética , Cartilla de ADN , Dermatán Sulfato/química , Disacáridos/análisis , Exones/genética , Vectores Genéticos , Mastocitos/enzimología , Mastocitos/ultraestructura , Ratones , Microscopía Electrónica , Reacción en Cadena de la Polimerasa , Bazo/enzimología , Sulfotransferasas/genética
8.
Eur J Immunol ; 40(5): 1296-302, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20186877

RESUMEN

The strength of interaction between the antigenic peptide-loaded MHC (MHC/p) and the TCR determines T-cell fate in the thymus. A high avidity interaction between the TCR and the MHC/p induces apoptosis of self-reactive T cells (negative selection), whereas a moderate avidity interaction rescues thymocytes from apoptosis and permits further differentiation to mature T cells (positive selection). Leukocyte common antigen-related molecule (LAR), a receptor-like protein tyrosine phosphatase, is expressed on immature thymocytes, but its role in thymocyte differentiation has not yet been fully elucidated. We analyzed LAR-deficient mice and demonstrated that LAR deficiency affected the differentiation and expansion of immature thymocytes as well as positive and negative selection. Furthermore, LAR deficiency resulted in a lower Ca2+ response. The results indicate that LAR is an important modulator of TCR signaling that controls thymocyte differentiation.


Asunto(s)
Linfopoyesis/fisiología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/fisiología , Subgrupos de Linfocitos T/citología , Animales , Antígenos CD/análisis , Supresión Clonal/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Receptores de Antígenos de Linfocitos T/inmunología
9.
J Gen Virol ; 91(Pt 4): 938-48, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20007353

RESUMEN

Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptor-binding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) alpha2,6galactose (Gal) or a Neu5Acalpha2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Acalpha2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Acalpha2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galbeta1,3/4GlcNAcalpha2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The alpha2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Acalpha2,3Gal more abundantly than Neu5Acalpha2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) alpha2,6Gal and Neu5Acalpha2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Receptores Virales/fisiología , Sustitución de Aminoácidos , Animales , Línea Celular , Embrión de Pollo/virología , Perros , Eritrocitos/química , Pruebas de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Ratones , Ácido N-Acetilneuramínico/sangre , Ácido N-Acetilneuramínico/química , Ratas , Ovinos , Porcinos
10.
Biol Pharm Bull ; 33(10): 1698-703, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20930378

RESUMEN

The present study examined modifications of ß-naphthoflavone (ß-NF)-induced cytochrome P450 1A1 (CYP1A1) expression by flavonoids in mouse hepatocytes in primary culture. Some flavonoids (apigenin, chrysin, flavone, flavanone, galangin, luteolin, and naringenin) by themselves induced CYP1A1 mRNA expression, especially flavone which was even more effective than ß-NF. The effect on ß-NF-induced CYP1A1 mRNA expression was varied, namely additive, suppressive, or both. An additive effect was observed after combined treatment with flavanone, naringenin, and chrysin, whereas kaempferol, myricetin, and quercetin decreased CYP1A1 levels. Apigenin, chrysin, galangin, luteolin, and morin synergistically enhanced ß-NF-induced CYP1A1 expression at 24 h, but considerably suppressed it at 9 h. The structure-activity relationship of flavonoids affecting CYP1A1 expression as inducers or inhibitors is discussed. The present observations suggest the need to reveal the mechanism by which CYP1A1 expression is modified by flavonoids for risk assessment, since CYP1A1 activates environmental carcinogenic polycyclic hydrocarbons and flavonoids are major constituents in food.


Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Flavonoides/farmacología , Hepatocitos/efectos de los fármacos , beta-naftoflavona/farmacología , Animales , Línea Celular , Citocromo P-450 CYP1A1/genética , Sinergismo Farmacológico , Flavonas/farmacología , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Relación Estructura-Actividad
11.
Biochim Biophys Acta ; 1780(4): 687-95, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18237557

RESUMEN

Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with 35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.


Asunto(s)
Células de la Médula Ósea/metabolismo , Sulfatos de Condroitina/biosíntesis , Mastocitos/metabolismo , Sulfotransferasas/metabolismo , Cloruro de Amonio/farmacología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Cloroquina/farmacología , Sulfatos de Condroitina/química , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Disacáridos/análisis , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Concentración de Iones de Hidrógeno , Macrólidos/farmacología , Mastocitos/citología , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfotransferasas/genética
12.
Biochem Biophys Res Commun ; 387(3): 575-80, 2009 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-19616512

RESUMEN

Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni(2+) affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc and Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.


Asunto(s)
Bombyx/genética , Receptores KIR2DL1/biosíntesis , Proteínas Recombinantes/farmacología , Animales , Baculoviridae/genética , Carbohidratos/inmunología , ADN/genética , Hemolinfa/inmunología , Humanos , Receptores KIR2DL1/genética , Receptores KIR2DL1/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología
13.
Glycoconj J ; 26(4): 433-43, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-18853253

RESUMEN

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to alpha2-->6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the alpha2-->3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of alpha2-->3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of alpha2-->6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.


Asunto(s)
Adaptación Biológica , Amnios/citología , Aves/virología , Membrana Corioalantoides/citología , Virus de la Influenza A/metabolismo , Óvulo/citología , Polisacáridos/análisis , Amnios/metabolismo , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Embrión de Pollo , Membrana Corioalantoides/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Datos de Secuencia Molecular , Polisacáridos/química
14.
Glycobiology ; 18(2): 145-51, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18056652

RESUMEN

We herein report N-glycosylation profiles of the individual tissues derived from the ascidian Ciona intestinalis. Multidimensional HPLC mapping revealed that the C. intestinalis expresses high-mannose-type oligosaccharides as major N-glycans, along with paucimannose-type and complex-type oligosaccharides, in a tissue-specific manner. Notably, the trimannosyl core carrying beta1,2-xylose and alpha1,3-fucose residues was identified as a principal N-glycan in the neural complex. As far as we know, this is the first description of xylosyl N-glycan expressed in deuterostome. Furthermore, we found that this xylosyl N-glycan is exclusively displayed on a membrane-associated protein so far described as a putative protein whose gene expression is specific for the neural complex. These data suggested that the xylosyl N-glycan is associated with some neural functions of C. intestinalis.


Asunto(s)
Ciona intestinalis/metabolismo , Polisacáridos/metabolismo , Xilosa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Ciona intestinalis/embriología , Embrión no Mamífero/metabolismo , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Glicosilación , Espectrometría de Masas , Datos de Secuencia Molecular , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Polisacáridos/química
15.
FEBS J ; 285(9): 1611-1634, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29542865

RESUMEN

The rapidly evolvable influenza A virus has caused pandemics linked to millions of deaths in the past century. Influenza A viruses are categorized by H (hemagglutinin; HA) and N (neuraminidase; NA) proteins expressed on the viral envelope surface. Analyses of past pandemics suggest that the HA gene segment comes from a nonhuman virus, which is then introduced into an immunologically naïve human population with potentially devastating consequences. As a prerequisite for infection, the nonhuman HA molecules of H1-H16 viruses must be able to bind to specific sialyl receptors on the host cell surface along the human respiratory tract. Thus, additional insight into the structures of host cell glycans and how different HAs interact with different glycans might provide new insight into the mechanisms underlying sustained infection and transmission in humans. In this work, we identified the sialyl N-glycans found in normal human alveoli and characterized the influenza viruses that preferentially bound to these different structures. We also determined the amino acid changes in HA that were linked to a switch of receptor-binding preference from nonhuman to pandemic, as well as pandemic to seasonal. Our data provide insight into why seasonal viruses are associated with reduced alveolar infection and damage and suggest new considerations for designing anti-HA vaccines and drugs. The results provide a better understanding of viral tropism and pathogenesis in humans that will be important for prediction and surveillance of zoonotic, pandemic, and epidemic influenza outbreaks. DATABASE: The novel hemagglutinin nucleotide sequences reported here were deposited in GISAID under the accession numbers of EPI685738 for A/Yamaguchi/20/2006(H1N1) and EPI685740 for A/Kitakyushu/10/2006(H1N1).


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/fisiología , Gripe Aviar/virología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/virología , Polisacáridos/fisiología , Enfermedades de las Aves de Corral/virología , Alveolos Pulmonares/patología , Receptores Virales/química , Tropismo Viral/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Unión Competitiva , Secuencia de Carbohidratos , Brotes de Enfermedades , Perros , Patos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Gripe Aviar/patología , Gripe Humana/epidemiología , Gripe Humana/patología , Células de Riñón Canino Madin Darby , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/patología , Pandemias , Polisacáridos/química , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/patología , Unión Proteica , Alveolos Pulmonares/química , Alveolos Pulmonares/virología , ARN Viral/genética , Estaciones del Año , Ácidos Siálicos/química , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/patología , Enfermedades de los Porcinos/virología , Replicación Viral , Zoonosis
16.
Cytometry A ; 71(12): 1003-10, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17972305

RESUMEN

Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single-cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live-cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope-based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen-specific B-cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus-neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody-based therapeutics and diagnosis for infectious diseases such as hepatitis viruses.


Asunto(s)
Linfocitos B/inmunología , Análisis por Micromatrices/métodos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Linfocitos B/citología , Citometría de Flujo/métodos , Hepatitis B/inmunología , Hepatitis B/virología , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/sangre , Vacunas contra Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Activación de Linfocitos , Ratones , Ratones SCID , Ratones Transgénicos , Análisis por Micromatrices/instrumentación
17.
Sci Rep ; 7(1): 1409, 2017 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-28469195

RESUMEN

Recombinant proteins produced in insect cells and insects, unlike those produced in mammalian cells, have pauci-mannose-type N-glycans. In this study, we examined complex-type N-glycans on recombinant proteins via coexpression of human ß-1,2-N-acetylglucosaminyltransferase II (hGnT II) and human ß1,4-galactosyltransferase (hGalT I) in silkworm pupae, by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The actin A3 promoter from B. mori and the polyhedrin promoter from Autographa californica multiple nucleopolyhedroviruses (AcMNPVs) were used to coexpress hGnT II and hGalT I. These recombinant BmNPVs were coexpressed with human IgG (hIgG), hGnT II and hGalT I in silkworm pupae. When hIgG was coexpressed with hGnT II, approximately 15% of all N-glycans were biantennary, with both arms terminally modified with N-acetylglucosamine (GlcNAc). In contrast, when hIgG was coexpressed with both hGnT II and hGalT I under the control of the polyhedrin promoter, 27% of all N-glycans were biantennary and terminally modified with GlcNAc, with up to 5% carrying one galactose and 11% carrying two. The obtained N-glycan structure was dependent on the promoters used for coexpression of hGnT II or hGalT I. This is the first report of silkworm pupae producing a biantennary, terminally galactosylated N-glycan in a recombinant protein. These results suggest that silkworms can be used as alternatives to insect and mammalian hosts to produce recombinant glycoproteins with complex N-glycans.


Asunto(s)
Glicosiltransferasas/biosíntesis , Animales , Bombyx , Vectores Genéticos , Glicosiltransferasas/química , Glicosiltransferasas/genética , Humanos , Nucleopoliedrovirus/genética , Polisacáridos/biosíntesis , Polisacáridos/química , Regiones Promotoras Genéticas , Pupa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
18.
J Chromatogr A ; 946(1-2): 291-4, 2002 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-11873977

RESUMEN

A sensitive method for the simultaneous determination of phenolic xenoestrogens such as bisphenol A, 2,4-dichlorophenol, 4-tert,-butylphenol, 4-n2-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-octylphenol, 4-nonylphenol was developed using reversed-phase LC and coulometric-array detection. Stepwise gradient elution with phosphoric acid in water-acetonitrile was used. The calibration curves were linear in the range of 5.0 (or 10.0)-1000 ng ml(-1) with correlation coefficients of 0.9978-0.9999, the limits of detection were 0.01-0.02 ng ml(-1). Sample clean-up was performed by solid-phase extraction (SPE) using 3M Empore extraction disks. Three commercial sorbents, C18, SDB-XD (styrene-divinylbenzene polymer) and SDB-RPS (sulfonated styrene-divinylbenzene polymer) were compared. The highest recoveries were obtained with SDB-RPS. They were above 70% with a relative standard deviation of less than 15%. The proposed method was applied to the determination of phenolic xenoestrogens in various water samples.


Asunto(s)
Cromatografía Liquida/métodos , Estrógenos no Esteroides/análisis , Fenoles/análisis , Contaminantes Químicos del Agua/análisis , Electroquímica , Sensibilidad y Especificidad
19.
Transpl Immunol ; 31(1): 48-53, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24867127

RESUMEN

BACKGROUND: N-glycans isolated from neonatal porcine islet-like cell clusters (NPCCs) were analyzed by a mapping technique, to examine the differences in glycosylation and antigenicity between adult pig islets (APIs) and NPCCs. METHODS: NPCCs were isolated from 1-to-3 day-old neonatal wild-type pigs and cultured for 9 days, using the technique described by Korbutt et al. The extract was proteolyzed by treatment with a chymotrypsin and trypsin mixture and further digested with glycoamidase A to release the N-glycans. After the removal of the peptide materials, the reducing ends of the N-glycans were derivatized with 2-aminopyridine. This mixture was applied to DEAE, amide and ODS columns. PA-oligosaccharides were also subjected to MALDI TOF-MS analysis. RESULTS: The NPCC glycans were comprised of 14 neutral, 5 mono-sialyl and 5 di-sialyl glycans. As a feature of the N-glycans of NPCC, NPCC contained large amounts of high mannose structures. On the other hand, all of the hybrid and complex types contained a Fucα1-6GlcNAc structure, but were not modified with sulfate residues. Among them, the NPCC preparation contained five neutral and two mono-sialyl glycans and two di-sialyl glycans that were not typically found in adult islets, and seven of these nine were not detected in human islets. Moreover, most of the structures could be clearly identified in this study. CONCLUSIONS: The data herein will be helpful for future studies of the glycoantigen associated with NPCC.


Asunto(s)
Islotes Pancreáticos/química , Polisacáridos/análisis , Animales , Animales Recién Nacidos , Cromatografía Líquida de Alta Presión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos
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