RESUMO
O-GlcNAcylation is a nutrient-driven post-translational modification known as a metabolic sensor that links metabolism to cellular function. Recent evidences indicate that the activation of O-GlcNAc pathway is a potential pro-survival pathway and that acute enhancement of this response is conducive to the survival of cells and tissues. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-d-pyranoside (SalA-4g), is a salidroside analogue synthesized in our laboratory by chemical structure-modification, with a phenyl ring containing a para-methoxy group and a sugar ring consisting of N-acetylglucosamine. We have previously shown that SalA-4g elevates levels of protein O-GlcNAc and improves neuronal tolerance to ischemia. However, the specific target of SalA-4g regulating O-GlcNAcylation remains unknown. To address these questions, in this study, we have focused on mitochondrial network homeostasis mediated by O-GlcNAcylation in SalA-4g's neuroprotection in primary cortical neurons under ischemic-like conditions. O-GlcNAc-modified mitochondria induced by SalA-4g demonstrated stronger neuroprotection under oxygen glucose deprivation and reoxygenation stress, including the improvement of mitochondrial homeostasis and bioenergy, and inhibition of mitochondrial apoptosis pathway. Blocking mitochondrial protein O-GlcNAcylation with OSMI-1 disrupted mitochondrial network homeostasis and antagonized the protective effects of SalA-4g. Collectively, these data demonstrate that mitochondrial homeostasis mediated by mitochondrial protein O-GlcNAcylation is critically involved in SalA-4g neuroprotection.
Assuntos
Acetilglucosamina/análogos & derivados , Metabolismo Energético , Isquemia/prevenção & controle , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/química , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Acetilglucosamina/farmacologia , Animais , Glucose/metabolismo , Glicosilação , Homeostase , Isquemia/metabolismo , Isquemia/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Oxigênio/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-DawleyRESUMO
(d)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis, a chronic and degenerative articular joint disease. N-acetyl-(d)-glucosamine, a compound that can be modified at the N position, is considered to improve the oral bioavailability of (d)-glucosamine and has been proven to possess greater in vitro chondroprotective activity compared with the parent agent. In this study, to further utilize these properties, we focus on the modification of the N position with a benzenesulfonyl and different isoxazole formyl groups. Among these compounds, the 3-(2-chlorobenzene)-5-methyl-isoxazole formyl chloride and p-methoxybenzenesulfonyl chloride modifying structures proved to be the most active of the series and efficiently processed the chondrocytes in vitro. These novel N-position substitution compounds may represent promising leads for osteoarthritis drug development.
Assuntos
Acetilglucosamina/análogos & derivados , Condrócitos/efeitos dos fármacos , Glucosamina/síntese química , Glucosamina/farmacologia , Acetilglucosamina/química , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Desenho de Fármacos , Glucosamina/química , Glicosaminoglicanos/metabolismo , Humanos , Estrutura Molecular , Osteoartrite/tratamento farmacológico , CoelhosRESUMO
(D)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis (OA), a chronic and degenerative articular joint disease. In our preceding paper, a series of novel O-6 phosphate N-acetyl (d)-glucosamine prodrugs aimed at improving the oral bioavailability of N-acetyl-(d)-glucosamine as its putative bioactive phosphate form were shown to have greater chondroprotective activity in vitro when compared to the parent agent. In order to extend the SAR studies, this work focuses on the O-3 and O-4 phosphate prodrugs of N-acetyl-(d)-glucosamine bearing a 4-methoxy phenyl group and different amino acid esters on the phosphate moiety. Among the compounds, the (l)-proline amino acid-containing prodrugs proved to be the most active of the series, more effective than the prior O-6 compounds, and well processed in chondrocytes in vitro. Data on human cartilage support the notion that these novel O-3 and O-4 regioisomers may represent novel promising leads for drug discovery for osteoarthritis.
Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosamina/síntese química , Cartilagem Articular/efeitos dos fármacos , Compostos Organofosforados/síntese química , Osteoartrite/tratamento farmacológico , Pró-Fármacos/síntese química , Acetilglucosamina/farmacologia , Agrecanas/metabolismo , Animais , Cartilagem Articular/metabolismo , Bovinos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Estabilidade de Medicamentos , Glicosaminoglicanos/metabolismo , Cobaias , Meia-Vida , Humanos , Técnicas de Cultura de Órgãos , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The swine influenza virus (H1N1) 2009 pandemic highlights the importance of having effective anti-viral strategies. Recently, oseltamivir (Tamiflu) resistant influenza viruses are identified; which further emphasizes the urgency in developing new antiviral agents. In influenza virus replication cycle, viral surface glycoprotein, hemagglutinin, is responsible for viral entry into host cells. Hence, a potentially effective antiviral strategy is to inhibit viral entry mechanism. To develop novel antiviral agent that inhibits viral entry, we analyzed 20,000 traditional Chinese medicine (TCM) ingredients in hemagglutinin subtype H1 sialic acid binding site found on H1N1 virus. We then performed molecular dynamics simulations to investigate receptor-ligand interaction of the candidates obtained from docking. Here, we report three TCM derivatives that have high binding affinities to H1 sialic acid binding site residues based on structure-based calculations. The top three derivatives, xylopine_2, rosmaricine_14 and rosmaricine_15, all have an amine group that interact with Glu83 and a pyridinium group that interact with Asp103. Molecular dynamics simulations show that these derivatives form strong hydrogen bonding with Glu83 but interact transiently with Asp103. We therefore suggest that an enhanced hemagglutinin inhibitor, based on our scaffold, should be designed to bind both Glu83 and Asp103 with high affinity.
Assuntos
Antivirais/química , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos , Vírus da Influenza A Subtipo H1N1/química , Medicina Tradicional Chinesa , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sequência de Aminoácidos , Antivirais/metabolismo , Sítios de Ligação , Hemaglutininas/química , Hemaglutininas/metabolismo , Humanos , Ligação de Hidrogênio , Vírus da Influenza A Subtipo H1N1/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Ligação Proteica , Alinhamento de SequênciaRESUMO
Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -beta1-4GlcA-alpha1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-D-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect. 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.
Assuntos
Acetilglucosamina/análogos & derivados , Desoxiglucose/análogos & derivados , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glucosamina/análogos & derivados , Heparitina Sulfato/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acetilglucosamina/farmacologia , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Glucosamina/farmacologia , Humanos , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
We demonstrated the prebiotic effect of lacto-N-biose I (Galbeta1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.
Assuntos
Acetilglucosamina/análogos & derivados , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/crescimento & desenvolvimento , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Animais , Colostro/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Lactente , GravidezRESUMO
BACKGROUND: Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal disease caused by deficiency of aspartylglucosaminidase. A thalamic T2 signal intensity decrease is associated with lysosomal diseases. PURPOSE: To investigate thalamic signal intensity in AGU by performing a retrospective review of brain magnetic resonance (MR) imaging studies of AGU patients. MATERIAL AND METHODS: A total of 25 MR examinations were available for 11 patients aged between 3 and 32 years (four patients underwent bone marrow transplantation). Of these, 13 examinations were performed after bone marrow transplantation. Five patients had from two to six examinations, and six patients had one examination each. In every patient, the diagnosis of AGU was confirmed by blood and urine tests. Eighteen examinations were performed with a 1.0T imager including dual spin-echo T2 and proton density (PD) axial and coronal images, and 10 examinations also included T1-weighted images. Seven examinations were performed with a 1.5T imager including turbo spin-echo axial and coronal T2-weighted images and axial fluid-attenuated inversion recovery (FLAIR) images; three examinations included T1-weighted three-dimensional magnetization-prepared rapid acquisition gradient-echo (3D MPRAGE) images. The signal intensity of the thalamus and pulvinar in every sequence was compared to that of the putamina. RESULTS: In AGU, thalamic alterations were first detectable on T2-weighted images (25 examinations in 11 patients) from the age of 3 years 6 months, showing decreased signal intensity in 21 of 24 examinations. T1-weighted images (13 examinations) showed slightly increased thalamic signal intensity in five out of seven examinations from the age of 7 years, and PD images (19 examinations) showed decreased signal intensity from the age of 16 years (three examinations). The pulvinar showed decreased signal intensity on spin-echo T2-weighted images for 14 of 18 examinations or on FLAIR sequences for seven of seven examinations from the age of 6 years and 6 months, both in patients with and without bone marrow transplantation, but no pulvinar alterations were observable on T1 and PD images. CONCLUSION: In AGU, the thalamus is affected. Pulvinar changes are visible only on T2-weighted images, and these may be the first changes reported in the group of lysosomal diseases.
Assuntos
Acetilglucosamina/análogos & derivados , Doenças por Armazenamento dos Lisossomos/diagnóstico , Doenças por Armazenamento dos Lisossomos/urina , Imageamento por Ressonância Magnética/métodos , Pulvinar/patologia , Acetilglucosamina/sangue , Acetilglucosamina/deficiência , Acetilglucosamina/urina , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Imageamento Tridimensional , Masculino , Estudos Retrospectivos , Tálamo/patologiaRESUMO
In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, beta-amyloid (Abeta), in Alzheimer's disease. The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan, part of which is protein and the remainder a specific linear polysaccharide, which is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of Alzheimer's disease, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it affects its amyloid-inducing properties. Since our previous report, 22 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, 12 soluble compounds have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures. In addition, one anomer of a 4-deoxy-d-glucosamine analog, which possesses AA-amyloid inhibitory properties in vivo is in the process of being assessed for its anti-Abeta activity using a murine transgenic model of brain Abeta amyloidogenesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified at the 2-N, C-3, C-4, C-3 and C-4, or C-6 positions. One compound modified at the 2-N position (QS231), which inhibits HS synthesis in hepatocyte cultures, has shown marked enhancing properties vis-à-vis AA amyloid deposition in vivo. Very instructive results with regard to HS structure and its relation to AA amyloid deposition should be forthcoming from analyses of the AA-associated HS generated with this compound.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Glicosaminoglicanos/farmacologia , Proteoglicanas de Heparan Sulfato/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acetilglucosamina/uso terapêutico , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Glicosaminoglicanos/síntese química , Glicosaminoglicanos/uso terapêutico , Proteoglicanas de Heparan Sulfato/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/uso terapêutico , Conformação ProteicaRESUMO
A 21-year-old right-handed man with definite diagnosis of aspartylglucosaminuria (AGU) presented with a 5-year history of progressive severe gait disturbance with frequent falls and generalized epileptic seizures triggered by unexpected stimuli. At one time, he was confined to a wheelchair because of the frequent falls. Electromyogram recording showed a large, excessive and not habituating motor startle response, with the classical and stereotyped order of muscle recruitment. During video-polygraphic recording, we recorded a reflex generalized tonic seizure triggered by a loud, unexpected acoustic stimulus. Brain magnetic resonance (MR) revealed no structural abnormality. A diagnosis of abnormal startle and startle epilepsy (SE) was made. The addition of clonazepam to valproate and phenobarbital led to a dramatic improvement in his abnormal startle and SE, and the patient was able to walk alone unaided. This report illustrates, for the first time, that abnormal startle and SE may occur in AGU and complicate its clinical picture. Recognition of this entity in AGU is important, as progressive gait disorder with frequent falls could be easily misinterpreted as an additional irreversible manifestation of the ongoing neurological deterioration characteristic of AGU.
Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosamina/urina , Epilepsia/etiologia , Epilepsia/fisiopatologia , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/complicações , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/fisiopatologia , Reflexo de Sobressalto/genética , Estimulação Acústica , Adulto , Anticonvulsivantes/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Diagnóstico Diferencial , Eletroencefalografia , Eletromiografia , Epilepsia/tratamento farmacológico , Transtornos Neurológicos da Marcha/etiologia , Humanos , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/metabolismo , Imageamento por Ressonância Magnética , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Reflexo de Sobressalto/efeitos dos fármacos , Resultado do TratamentoRESUMO
In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, e.g., amyloid beta (Abeta) in Alzheimer's disease (AD). The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan (HSPG), part of which is protein, and the remainder is a specific linear polysaccharide that is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of AD, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it loses its amyloid-inducing properties. Since our previous report, 14 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, eight have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures, and the two anomers of a 4-deoxy-D-glucosamine analog have been assessed for their inflammation-associated amyloid (AA amyloid) inhibitory properties in vivo. The promising in vivo results with these two compounds have prompted studies using a murine transgenic model of brain Abeta amyloidogenesis. A macrophage tissue-culture model of AA amyloidogenesis has been devised based on the work of Kluve-Beckerman et al. and modified so as to assess compounds in the absence of potential in vivo confounding variables. Preliminary results indicate that the anomers of interest also inhibit AA amyloid deposition in macrophage tissue culture. Finally, an in vitro technique, using liver Golgi (the site of HS synthesis) rather than whole cells, has been devised to directly assess the effect of analogs on HS biosynthesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified either at the 2-N, C-3, C-4, or C-3 and C-4 positions. Results with the majority of the 2-N analogs suggest that hepacyte N-demethylases remove the N-substituent removal. Several of these have the desired effect on HS biosynthesis using hepatocyte cultures and will be assessed in the culture and in vivo AA amyloid models. To date 3-deoxy and 3,4-dideoxy analogs have failed to affect HS synthesis significantly. Compounds incorporating the 6-deoxy structural feature are currently being designed and synthesized.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Membrana Basal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Proteoglicanas de Heparan Sulfato/antagonistas & inibidores , Placa Amiloide/efeitos dos fármacos , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acetilglucosamina/uso terapêutico , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Animais , Membrana Basal/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Glicosaminoglicanos/uso terapêutico , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , Hepatócitos , Macrófagos , Camundongos , Estrutura Molecular , Placa Amiloide/metabolismoRESUMO
N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1 cDNA. In this paper, we report the cloning, characterization and chromosomal mapping of mouse GalNAc4ST-1 and GalNAc4ST-2. Mouse GalNAc4ST-1 and GalNAc4ST-2 contain single open reading frames that predict type II transmembrane proteins composed of 417 and 413 amino acid residues, respectively. The amino acid sequence identity between the two isoforms is 49%. When the cDNA was transfected to COS-7 cells, sulfotransferase activities toward carbonic anhydrase VI and GalNAcbeta1-4GlcNAcbeta1-octyl were overexpressed, but the sulfotransferase activity toward chondroitin showed no increase over the control level. Northern blot analysis showed that the 2.4 kb messages of GalNAc4ST-1 and GalNAc4ST-2 were strongly expressed in the kidney, where both of the human isoforms were hardly expressed. Reverse transcription-PCR analysis showed that, unlike human GalNAc4ST-1, the expression of mouse GalNAc4ST-1 in the pituitary gland was only marginal, while that of GalNAc4ST-2 in the pituitary gland was as high as that in the kidney. These results suggest that the functions of the two GalNAc4ST isoforms may differ between human and mouse. By fluorescence in situ hybridization, the GalNAc4ST-1 and GalNAc4ST-2 genes were localized to mouse chromosome 7B3 distal-B5 proximal and chromosome 18A2 distal-B1 proximal, respectively.
Assuntos
Acetilgalactosamina/metabolismo , Acetilglucosamina/metabolismo , Cromossomos/enzimologia , Dissacarídeos/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilglucosamina/análogos & derivados , Sequência de Aminoácidos , Aminoácidos/química , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Humanos , Isoenzimas , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfatos/metabolismo , Distribuição TecidualRESUMO
The Galbeta1-->4(SO(3)(-)-->6)GlcNAc moiety is present in various N-linked and O-linked glycans including keratan sulfate and 6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found beta1,4-galactosyltransferase (beta4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata, K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the partial amino acid sequence of the enzyme revealed that the purified beta4GalT has a similar sequence to human beta4GalT-IV. To confirm this result, we prepared cDNA for each of the seven beta4GalTs cloned to date and examined substrate specificities using the membrane fractions derived from beta4GalT-transfected COS-7 cells. When using several N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only beta4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Galbeta1-->3(SO(3)(-)-->6GlcNAcbeta1-->6) GalNAcalpha1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that beta4GalT-IV is a 6SGN-specific beta4GalT and may be involved in the biosynthesis of various glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety.
Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Esfingosina/análogos & derivados , Aminas/química , Sequência de Aminoácidos , Aminoácidos/química , Amino Açúcares/química , Animais , Northern Blotting , Células COS , Sequência de Carboidratos , Cromatografia , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Humanos , Sulfato de Queratano/metabolismo , Antígenos CD15/química , Antígenos CD15/metabolismo , Lipídeos , Linfonodos/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Peptídeos/química , Polissacarídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Sefarose/farmacologia , Esfingosina/química , Suínos , Distribuição Tecidual , Transfecção , Difosfato de Uridina/metabolismoRESUMO
A new phosphorus-containing compound (1) was detected by (31)P NMR spectroscopy in Streptomyces sp. A50. Compound 1, 1(alpha)-O-methyl-2-(N-acetyl)glucoseamine-6-O-phosphate-1(alpha)-2-(N-acetyl)glucosamine, exhibited a pK(a) value around zero. The compound was found both in the extracellular culture broth and in the cells. While very low concentrations of 1 were found in the culture broth of other species of Streptomyces, its presence in high concentrations was specific to Streptomyces sp. A50. The highly acidic compound was isolated from the broth, and its structure was elucidated by a combination of 1D-, 2D-homonuclear, and inverse heteronuclear NMR techniques and mass spectroscopy.
Assuntos
Acetilglucosamina/química , Acetilglucosamina/metabolismo , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Streptomyces/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/análise , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/análise , Espectrometria de Massas , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fósforo/química , Fósforo/metabolismoRESUMO
Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.
Assuntos
Fabaceae/genética , Lectinas/síntese química , Lectinas/genética , Proteínas de Plantas/síntese química , Proteínas de Plantas/genética , Plantas Medicinais , Acetilglucosamina/análogos & derivados , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Sequência de Aminoácidos , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Sítios de Ligação/genética , Fabaceae/química , Vetores Genéticos/síntese química , Lectinas/biossíntese , Lectinas/metabolismo , Manose/genética , Manose/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Lectinas de Plantas , Proteínas de Plantas/biossíntese , Proteínas de Plantas/metabolismo , Engenharia de Proteínas , Albumina Sérica/genética , Albumina Sérica/metabolismo , Soroalbumina Bovina/genética , Soroalbumina Bovina/metabolismo , Vírion/genética , Vírion/metabolismoAssuntos
Acetilglucosamina/análogos & derivados , Antifúngicos/farmacologia , Quitina/metabolismo , Compostos de Dansil/metabolismo , Corantes Fluorescentes/metabolismo , Acetilglucosamina/síntese química , Acetilglucosamina/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Parede Celular/efeitos dos fármacos , Quitina Sintase/antagonistas & inibidores , Compostos de Dansil/síntese química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Nucleosídeos de Pirimidina/farmacologiaRESUMO
Effects of two inhibitors, allosamidin and (2-acetamido-2-deoxy-D-glucopyranosylidene)amino phenylcarbamate (PUGNAC), have been assessed on chitinolytic activities of two plants, Pinus sylvestris L. and Eucalyptus pilularis Sm., and of seven fungi. Pinus sylvestris and E. pilularis root endochitinase activities were inhibited by allosamidin. Activities of P. sylvestris were more sensitive to inhibition than those of E. pilularis. The mechanism of inhibition varied with the plant species and the enzyme involved. PUGNAC inhibited beta-N-acetylglucosaminidase and exochitinase activities in root extracts from both plant species. In all cases PUGNAC acted as a reversible competitive inhibitor. Both inhibitors also affected chitinolytic activities from the fungi screened. Allosamidin inhibited endochitinase activities from both the mycorrhizal and pathogenic fungi tested. In addition, exochitinase activity from the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. was inhibited by allosamidin. PUGNAC inhibited beta-N-acetylglucosaminidase activity from all the fungi tested. PUGNAC was also a potent inhibitor of both exo- and endochitinase activities from the fungi, except P. involutus. Competitive inhibition was the most common form. These findings show allosamidin does inhibit endochitinase activity in plants and the ability of PUGNAC to inhibit not only beta-N-acetylglucosaminidase activity but also fungal endochitinase activity may be useful to distinguish between host and fungal endochitinase activities in symbiotic or pathogenic dual systems.
Assuntos
Acetilglucosamina/análogos & derivados , Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fungos/enzimologia , Oximas/farmacologia , Fenilcarbamatos , Árvores/enzimologia , Árvores/microbiologia , Trissacarídeos/farmacologia , Acetilglucosamina/farmacologia , Sequência de Carboidratos , Eucalyptus/enzimologia , Cinética , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Plantas Medicinais , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Adhesion of A-121 human ovarian carcinoma cells to extracellular matrix is partly mediated via interaction between galaptin, an endogenous beta-galactoside-binding lectin present in extracellular matrix, and specific cell surface carbohydrate receptors identified as lysosomal associated membrane proteins, lamp-1 and lamp-2. In this study, we report that adhesion of human ovarian carcinoma cells to polystyrene plates coated with polymerized human splenic galaptin can be inhibited by polyclonal antibodies raised against lamp-1 and lamp-2 molecules and by pretreatment of A-121 human ovarian carcinoma cells with glucosamine analogs: 2-acetamido-1,4,6-tri-O-acetyl-3- deoxy-3-fluoro-alpha-D-glucopyranose (3-F-GlcNAc) and 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-alpha-D-glucopyranose (4-F-GlcNAc). A 48-h exposure of A-121 cells to individual sugar analogs, or to a combination of the two, resulted in a concentration-dependent inhibition of cellular attachment to polymerized galaptin. Both drugs inhibited glycoprotein biosynthesis as measured by cellular incorporation of labeled [3H]glucosamine and [3H]fucose with negligible effects on [3H]thymidine and [3H]leucine incorporation and cell growth. As a result of drug action on glycoprotein biosynthesis, an alteration in the structure of the galaptin receptor was noted by indirect immunofluorescence and Western blot analysis. Moreover, probing gels of cell extracts with anti-lamp antibodies or Datura stramonium lectin demonstrated significant changes in the reactivity and pattern of glycoprotein staining, suggesting an effect of sugar analogs on the glycosylation of various cellular receptor molecules. The greatest change was observed when tumor cells were exposed to a combination of the two sugar analogs. These studies suggest that specific endogenous lectins and their surface receptors play a role in tumor cell adhesion and perhaps metastasis and may serve as suitable targets for therapeutic exploitation.
Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Antígenos CD , Adesão Celular/fisiologia , Hemaglutininas/fisiologia , Lectinas/fisiologia , Neoplasias Ovarianas/fisiopatologia , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Datura stramonium , Matriz Extracelular/fisiologia , Feminino , Imunofluorescência , Galectina 1 , Humanos , Cinética , Lectinas/farmacologia , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/fisiologia , Neoplasias Ovarianas/patologia , Lectinas de Plantas , Plantas Medicinais , Plantas Tóxicas , Baço , Células Tumorais CultivadasRESUMO
Central mechanisms involved in feeding suppression produced by 1-deoxy-D-glucosamine (1-DGlcN) and 1-deoxy-N-acetylglucosamine (1-DGlcNAc) are unclear. To clarify the mechanisms, we investigated the role of hypothalamic neuronal histamine (HA) in feeding suppression induced by 1-DGlcN and 1-DGlcNAc in rats. Food intake was suppressed for 3 days after a single infusion of 24 mumol 1-DGlcN into the third cerebroventricle (i.c.v.). Depletion of presynaptic HA due to intraperitoneal infusion (i.p.) of alpha-fluoromethylhistidine (FMH), a specific inhibitor of the HA synthesizing enzyme histidine decarboxylase (HDC), abolished feeding suppression completely. Blockade of postsynaptic H1-receptors by i.p. injection of 26 mumol chlorpheniramine also abolished the suppression. Oral administration of 2.4 mmol 1-DGlcNAc suppressed food intake. However, depletion of neuronal HA due to FMH did not affect the suppression. I.c.v. infusion of 24 mumol 1-DGlcN increased turnover rate of HA at 1 h after the infusion. Hypothalamic HA concentration, but not that of tele-methylhistamine (t-MH), increased at 24 h after i.c.v. infusion of 1-DGlcN, which suggests a correlation between HA concentration and the behavioral response. These results indicate that 1-DGlcN, but not 1-DGlcNAc, modulates feeding suppression through HA neurons in the hypothalamus. Differences in mechanisms of feeding suppression by these aminoglucoses may depend on the principal sites of action in the brain and/or peripheral organs.
Assuntos
Acetilglucosamina/análogos & derivados , Depressores do Apetite/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Glucosamina/análogos & derivados , Histamina/fisiologia , Hipotálamo/fisiologia , Neurônios/fisiologia , Acetilglucosamina/farmacologia , Animais , Glicemia/metabolismo , Clorfeniramina/farmacologia , Glucosamina/farmacologia , Histamina/análogos & derivados , Histamina/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Histidina Descarboxilase/antagonistas & inibidores , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Ratos , Ratos WistarRESUMO
The occurrence of phosphate-containing sialyl oligosaccharides in bovine colostrum was investigated. Two major sialyl oligosaccharide phosphates were identified, one of which was structurally similar to the previously characterized sialyl oligosaccharide 1-phosphates of human urine. The second sialyl oligosaccharide phosphate of bovine colostrum was found to be of a novel type. Structural studies including monosaccharide and phosphate analysis, glycosidase and phosphatase treatments, methylation analysis, and periodate treatment indicated the structure of this compound to be NeuAc alpha 2-6Gal beta 1-4GlcNAc-6-P. This provides the first evidence for the occurrence of N-acetylglucosamine 6-phosphate as an integral component in complex carbohydrates.
Assuntos
Acetilglucosamina/análise , Colostro/análise , Glucosamina/análogos & derivados , Oligossacarídeos/isolamento & purificação , Acetilglucosamina/análogos & derivados , Fosfatase Alcalina , Animais , Sequência de Carboidratos , Bovinos , Cromatografia em Camada Fina , Escherichia coli/enzimologia , Feminino , Galactose/análise , Metilação , Neuraminidase , Fosforilação , Gravidez , beta-GalactosidaseRESUMO
An enzyme preparation from jack-bean meal hydrolyzed beta-aspartylglucosylamine linkages in glycopeptides. The enzyme could release sialic acid-containing complex-type oligosaccharides as well as high-mannose-type and hybrid-type oligosaccharides. The products were equimolar amounts of ammonia, oligosaccharide and peptide. The enzyme cleaved glycopeptides with three or more amino acid residues, whereas it did not hydrolyze GlcNAc-Asn. The mechanism of action of the enzyme and substrate specificity so far tested were similar to those of the glycopeptidase from almonds.