RESUMO
INTRODUCTION: Severe blood loss and related haemodilution during cardiac surgery result in a reduced platelet count which may lead to impaired primary haemostasis. Additionally, the reduced haematocrit lowers rheological forces in circulation and may account for lowered platelet adhesiveness and potentially reduced von Willebrand factor (VWF) activity. These mechanisms may lead to postoperative bleeding. Aim of this study was the examination of VWF activity and VWF-mediated platelet adhesion to collagen under conditions of haemodilution. MATERIALS AND METHODS: An in vitro flow chamber was utilized to examine the primary haemostasis under a high arterial shear rate of 1500s-1 at variable VWF concentrations, platelet counts and haematocrit levels. RESULTS: Under a high arterial shear rate, VWF activity is highly dependent on blood viscosity. Both VWF-collagen binding and VWF-mediated platelet adhesion to collagen were significantly increased with increasing haematocrit. Interestingly, we found slight differences in the VWF multimer sizes able to bind collagen under different shear stress conditions. Under conditions of haemodilution, platelet adhesion was strongly dependent on VWF concentration. Increasing VWF concentration improved platelet adhesiveness under low haematocrit conditions (30%) and variable platelet counts (80, 150 and 250×109/L). This effect was nearly abolished at very low platelet count levels of 50×109/L. CONCLUSIONS: VWF improves platelet function under conditions of haemodilution. Therefore, increasing VWF concentration may represent a complementary strategy to administration of platelet concentrates for the management of bleeding in thrombocytopenia.
Assuntos
Hemodiluição/métodos , Hemostasia/efeitos dos fármacos , Fator de von Willebrand/uso terapêutico , Humanos , Fator de von Willebrand/farmacologiaRESUMO
BACKGROUND: O-glycosylation of von Willebrand factor (VWF) affects many of its functions; however, there is currently no information on the occupancy of the 10 putative O-glycosylation sites. OBJECTIVES: The aim of this study was the site-specific analysis of VWF O-glycosylation. METHODS: Tryptic VWF-O-glycopeptides were isolated by lectin affinity chromatography and/or by reverse-phase high-performance liquid chromatography. Subsequently, the purified glycopeptides were analyzed by glycosidase digestion and mass spectrometry. RESULTS: We found that all 10 predicted O-glycosylation sites in VWF are occupied. The majority of the glycan structures on all glycosylation sites is represented by disialyl core 1 O-glycan. The presence of core 2 O-glycan was also confirmed; interestingly, this structure was not evenly distributed among all 10 glycosylation sites. Analysis of the glycopeptides flanking the A1 domain revealed that generally more core-2-type O-glycan was present on the C-terminal Cluster 2 glycopeptide (encompassing T(1468) , T(1477) , S(1486) and T(1487) ) compared with the N-terminal Cluster 1 glycopeptide (encompassing T(1248) , T(1255) , T(1256) and S(1263) ). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T(2298) in the C1 domain. In addition, we identify sulfation of core 2 O-glycans and the presence of the rare Tn antigen. CONCLUSIONS: This is the first study to describe the qualitative and semi-quantitative distribution of O-glycan structures on all 10 O-glycosylation sites, which will provide a valuable starting point for further studies exploring the functional and structural implications of O-glycosylation in VWF.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Polissacarídeos/química , Fator de von Willebrand/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Glicopeptídeos/química , Glicosídeo Hidrolases/química , Glicosilação , Humanos , Lectinas/química , Espectrometria de Massas , Lectinas de Plantas/química , Domínios Proteicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. OBJECTIVES: To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. PATIENTS/METHODS: Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3 µg kg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. RESULTS: Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factor VIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116 IU mL(-1) (interquartile range [IQR] 102-154 IU mL(-1) ) to a median of 160 IU mL(-1) (IQR 126-187 IU mL(-1) ) (P = 0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. In vitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. CONCLUSIONS: Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Desamino Arginina Vasopressina/uso terapêutico , Hemostáticos/uso terapêutico , Hemorragia Pós-Operatória/tratamento farmacológico , Idoso , Testes de Coagulação Sanguínea , Plaquetas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidilserinas/sangue , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Hemorragia Pós-Operatória/sangue , Hemorragia Pós-Operatória/etiologia , Resultado do Tratamento , Fator de von Willebrand/metabolismoRESUMO
The first meeting of international specialists in the field of von Willebrand disease (VWD) was held in the Åland islands in 1998 where Erik von Willebrand had first observed a bleeding disorder in some members of a family from Föglö and a summary of the meeting was published in 1999. The second meeting was held in 2010 and a report of the meeting was published in 2012. Topics covered included progress in understanding of VWD over the last 50 years; multimers; classification of VWD; pharmacokinetics and laboratory assays; genetics; treating the paediatric patient; prophylaxis; geriatrics; gene therapy and treatment guidelines. This third meeting held over 3 days covered the structure and function of von Willebrand factor (VWF); type 1 VWD, the most common form of the disease; a lifespan of pharmacokinetics in VWD; detecting inhibitors in VWD patients; and special challenges in understanding and treating the female VWD patient.
Assuntos
Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/terapia , Animais , Fator VIII/metabolismo , Feminino , Humanos , Doenças de von Willebrand/genética , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Complement factor H (CFH) acts as major regulator of the alternative pathway of complement and its mutations and polymorphisms predispose to various human diseases. We aimed to develop a scalable purification process of CFH from human plasma fractions to supply a pathogen-safe and functional CFH concentrate. MATERIALS AND METHODS: Starting with intermediates of cold ethanol fractionation of plasma, CFH purification was performed with three chromatographic steps including solvent detergent treatment and nanofiltration. CFH functionality was tested by a haemolysis assay using sheep erythrocytes, by determining decay acceleration activity on C3 convertases and cofactor activity of C3b cleavage. CFH identity was confirmed by Western blot and mass spectrometry. RESULTS: Three scalable chromatographic steps highly purified full-length and native CFH from human plasma fractions. The purification process enabled the removal of truncated and dysfunctional CFH species, yielding a native CFH concentrate as demonstrated in sensitive functional in vitro assays. CONCLUSION: This novel process provides a pathogen-safe and functional CFH concentrate that can be produced on an industrial scale and is suitable for pre-/clinical studies.
Assuntos
Fator H do Complemento/química , Fator H do Complemento/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração , Complemento C3/química , Complemento C3/genética , Complemento C3/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Humanos , Espectrometria de Massas , Polimorfismo Genético , Ligação Proteica , OvinosRESUMO
BACKGROUND AND OBJECTIVES: A high concentration of large size polymers in intravenous immunoglobulin preparations was always correlated with high anticomplementary activity (ACA). In former days, high ACA was also linked to adverse reactions in patients. The goal of this study was to scrutinize critical parameters of the ACA assay and the influence of different polymer variants of IgG on the complement consumption. MATERIALS AND METHODS: Critical reagents as the complement and the preparation of erythrocytes were investigated. The influence of molecular integrity of IgG on the ACA was tested by subjecting IgG solutions ranging from pH 4.5 to 7.0 to heat treatment at 60 degrees C. RESULTS: The different complement batches had a significant impact on the test result of the ACA assay. It was demonstrated that polymers, provoked by heat treatment at pH values above 5.5, consumed complement almost completely whereas a polymer content up to 12% formed under acidic conditions did not lead to an increase in ACA. CONCLUSION: It was shown that suitable complement batches have to be identified in a screening procedure. Furthermore, it could be demonstrated that IgG polymers formed in the neutral pH range during heat treatment were potential ACA inducing compounds. Manufacturing the IVIG preparations under acidic conditions may help to avoid the formation of those ACA active polymers. Thus, intensive analysis of ACA during process development and validation is recommended.
Assuntos
Artefatos , Ativação do Complemento , Imunoglobulina G/imunologia , Imunoglobulinas Intravenosas/efeitos adversos , Animais , Cromatografia em Gel , Proteínas do Sistema Complemento/química , Cobaias , Hemólise , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulinas Intravenosas/imunologia , Indicadores e Reagentes , Nefelometria e Turbidimetria , Reprodutibilidade dos Testes , OvinosRESUMO
BACKGROUND AND OBJECTIVES: The pathomechanism of thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS) is associated with a severe deficiency of ADAMTS13 and factor H. The aim of this study was to quantify the levels of ADAMTS13 and factor H in the pharmaceutically licensed plasma for transfusion, Octaplas, and the universally applicable plasma, Uniplas (development product, working title). Furthermore, Octaplas batches of blood groups A, B, O, AB, and plasmas derived from different sources were compared. MATERIALS AND METHODS: Twenty-four Octaplas and three Uniplas batches were selected for the study. ADAMTS13 activities were measured by fluorescence resonance energy transfer assay, ADAMTS13 antigen levels were quantified using enzyme-linked immunosorbent assay test kit, while factor H antigen levels were detected using radial immunodiffusion (RID) methods. In addition, von Willebrand factor (vWF) multimeric analyses were performed. RESULTS: Both Octaplas, produced from US and European plasma of different blood groups, and Uniplas contain ADAMTS13 antigen and activity levels as well as factor H concentrations at normal levels without significant differences. In addition, Octaplas and Uniplas show a vWF multimeric pattern comparable to normal plasma. CONCLUSION: The study revealed that Octaplas and Uniplas contain normal levels of ADAMTS13 at low batch-to-batch variations. Therefore, both products can substitute the missing or neutralized protease activity in TTP patients and thereby limit vWF-dependent (platelet-related) thrombosis. In addition, both plasma products contain factor H at a physiological level, and, thus can be used efficiently in the treatment of aHUS patients, which have been shown to benefit from plasma administration.
Assuntos
Proteínas ADAM/análise , Transfusão de Componentes Sanguíneos , Fator H do Complemento/análise , Plasma/química , Proteína ADAMTS13 , Síndrome Hemolítico-Urêmica/terapia , HumanosRESUMO
This study summarises the biochemical and functional properties of a new generation plasma-derived, double virus inactivated von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate, Wilate, targeted for the treatment of both von Willebrand disease (VWD) and haemophilia A. The manufacturing process comprises two chromatographic steps based on different performance principles, ensuring a high purity of the concentrate (mean specific activity in 15 consecutive production batches: 122 IU FVIII:C/mg total protein) and, thus, minimising the administered protein load to the patient (specification: < or = 15 mg total protein per 900 IU Wilate). The optimised solvent/detergent (S/D) treatment and prolonged terminal dry-heat (PermaHeat) treatment of the lyophilised product at a specified residual moisture (RM) provide two mechanistically independent, effective and robust virus inactivation procedures for enveloped viruses and one step for non-enveloped viruses. These process steps are aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules, as proven by state-of-the-art assays covering the diverse features of importance. The VWF multimeric pattern is close to the one displayed by normal plasma, with a consistent content of more than 10 multimers, but a relatively lower portion of the very high multimers. The multimeric triplet structure is normal, underlining the gentle and effective manufacturing process, which does not require the addition of protein stabilisers at any step. The balanced activity ratio of VWF to FVIII is close to that of plasma from healthy subjects, rendering Wilate suitable also for the safe and effective treatment of patients with VWD.
Assuntos
Fator VIII/isolamento & purificação , Fator VIII/fisiologia , HIV-1/fisiologia , Inativação de Vírus , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/fisiologia , Proteínas Sanguíneas/fisiologia , Fator VIII/química , HIV-1/isolamento & purificação , Humanos , Segurança , Fator de von Willebrand/químicaRESUMO
Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here.
Assuntos
Fatores de Coagulação Sanguínea/normas , Vitronectina/análise , Fatores de Coagulação Sanguínea/química , Fatores de Coagulação Sanguínea/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Dimerização , Contaminação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vitronectina/imunologia , Vitronectina/metabolismoRESUMO
The biochemical and immunochemical aspects of the development of inhibitors with a plasma-derived, double-virus inactivated factor VIII (FVIII) concentrate (marketed as Octavi SDPlus in Germany and Bisinact in Belgium) are described. A total of 12 cases of inhibitor formation (predominantly type II) were reported in Germany, 8 in Belgium but none in Portugal. Initially, the only difference between the non-pasteurised, SD virus-inactivated product Octavi and the pasteurised product Octavi SDPlus appeared to be pasteurisation, though subsequently, the quality of source material for the product was found to differ in different countries. Separation studies revealed the presence of a 40 kDa peptide fragment in some batches. It was subsequently shown that there was a strong correlation between inhibitor development and batches containing the 40 kDa marker, and a relationship between elevated markers of coagulation activation (FPA in particular) and the occurrence of the 40 kDa marker. Further work revealed that analytical methods commonly used for quality control were not suitable to highlight batch-to-batch differences. It was concluded that inhibitor potential (neoantigenicity) in Octavi SDPlus arose due to two effects; degradation of FVIII already present in source material; and heating of unstable FVIII degradation products. In this case, inhibitors were not caused by the overall production process, nor by GMP failures. The problem of inhibitor potential can be avoided if appropriate preventive measures are taken. Further work is needed to prove non-neoantigenicity and to reinforce the scientific findings, and to characterise pilot batches.
Assuntos
Fator VIII/imunologia , Cromatografia Líquida de Alta Pressão , Epitopos/efeitos adversos , Fator VIII/metabolismo , Humanos , Manufaturas , Peso Molecular , Plasma/químicaRESUMO
Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.
Assuntos
Adenosina Trifosfatases/química , Antígeno Carcinoembrionário/química , Moléculas de Adesão Celular/química , Fígado/química , Oligossacarídeos/química , Sialoglicoproteínas/química , Animais , Antígenos CD/química , Antígenos de Diferenciação/química , Sequência de Carboidratos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Wistar , Análise de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.
Assuntos
Manose/metabolismo , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células Cultivadas , Endocitose , Glicosilação , Hidrólise , Cinética , Glicoproteínas de Membrana/química , Peso Molecular , Ratos , Células Tumorais CultivadasRESUMO
The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.
Assuntos
Ativadores de Plasminogênio/metabolismo , Saliva/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Quirópteros , Cricetinae , Cricetulus , Glicopeptídeos/análise , Glicosilação , Dados de Sequência Molecular , Oligossacarídeos/análise , Mapeamento de Peptídeos , Ativadores de Plasminogênio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.
Assuntos
Fucose/análise , Ativadores de Plasminogênio/química , Saliva/enzimologia , Sequência de Aminoácidos , Animais , Células CHO , Bovinos , Quirópteros , Cromatografia Líquida de Alta Pressão , Cricetinae , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mapeamento de Peptídeos , Ativadores de Plasminogênio/isolamento & purificação , Ativadores de Plasminogênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Treonina , Ativador de Plasminogênio Tecidual/química , TransfecçãoRESUMO
Incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of different gangliosides of rat liver was examined. After intraperitoneal administration of 2-deoxy-D-galactose it was shown by GLC/MS analysis that this hexose analogue is metabolized and incorporated into all the gangliosides investigated, and predominantly into GM3 and GD3. In both of these gangliosides, 25-55% of the galactose residues were substituted by 2-deoxy-D-galactose. The epimer, 2-deoxy-D-glucose, was not detectable.
Assuntos
Galactose/análogos & derivados , Gangliosídeos/metabolismo , Animais , Sequência de Carboidratos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Gangliosídeo G(M3)/metabolismo , Galactose/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Injeções Intraperitoneais , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos BUF , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
The canalicular domain-specific glycoprotein gp110, which recently has been shown to function as an ATP-dependent taurocholate transporter, has been purified 1800-fold from rat liver plasma membranes. gp110 has been characterised as an integral plasma membrane protein with M(r) of 100,000-115,000 and pI of 2.5-3.5 and possesses a highly glycosylated and negatively charged extra-cellular domain. The broad range of M(r) and pI values results from the existence of numerous glycoforms composed of sialylated N-glycans. After deglycosylation, the polypeptide has M(r) 48,000 and pI 5.0. In primary cultures of rat hepatocytes, gp110 is synthesised with M(r) 110,000, while in the presence of tunicamycin the non-glycosylated form has M(r) 48,000. In the presence of 1-deoxymannojirimycin, two forms of M(r) 83,000 and M(r) 91,000 were found, which were converted by endo-beta-N-acetylglucosaminidase H into a single 52,000-M(r) band, indicating the existence of two basic glycoforms at the oligomannosyl stage of biosynthesis. gp110 was phosphorylated at serine residues in primary cultures of hepatocytes. The sequences of ten internal peptides of gp110 were identical to the sequence of the high-M(r) form of ecto-ATPase, but ecto-ATPase activity from plasma-membrane extracts was not depleted by anti-(gp110) serum. In contrast, Fab fragments of these antibodies inhibit the aggregation of freshly isolated hepatocytes.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/farmacologia , Canalículos Biliares/química , Proteínas de Transporte/isolamento & purificação , Fígado/química , Sequência de Aminoácidos , Animais , Canalículos Biliares/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Adesão Celular , Membrana Celular/química , Células Cultivadas , Glicosilação , Técnicas de Imunoadsorção , Ponto Isoelétrico , Fígado/ultraestrutura , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Ratos , Análise de Sequência , Ácido Taurocólico/metabolismoRESUMO
In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.
Assuntos
Glucosamina/análogos & derivados , Hexosaminas/metabolismo , Ácidos Siálicos/biossíntese , Animais , Radioisótopos de Carbono , Membrana Celular/metabolismo , Cromatografia em Papel , Cromatografia Gasosa-Espectrometria de Massas , Glucosamina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Pulmão/metabolismo , Masculino , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Ácidos Siálicos/isolamento & purificação , Relação Estrutura-Atividade , Glândula Submandibular/metabolismoRESUMO
The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.
Assuntos
Galactose/análogos & derivados , Glicoproteínas de Membrana/biossíntese , Trifosfato de Adenosina/metabolismo , Ascite/metabolismo , Radioisótopos de Carbono , Carcinoma Hepatocelular , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Fucose/metabolismo , Galactose/metabolismo , Humanos , Cinética , Neoplasias Hepáticas , Glicoproteínas de Membrana/isolamento & purificação , Polissacarídeos/biossíntese , Polissacarídeos/isolamento & purificação , Técnica de Diluição de Radioisótopos , Células Tumorais CultivadasRESUMO
Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.