Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Dermatitis Atópica/tratamiento farmacológico , Inmunoglobulina E/sangre , Adulto , Asma/sangre , Asma/tratamiento farmacológico , Asma/epidemiología , Asma/inmunología , Comorbilidad , Conjuntivitis Alérgica/sangre , Conjuntivitis Alérgica/tratamiento farmacológico , Conjuntivitis Alérgica/epidemiología , Conjuntivitis Alérgica/inmunología , Dermatitis Atópica/diagnóstico , Dermatitis Atópica/epidemiología , Dermatitis Atópica/inmunología , Femenino , Estudios de Seguimiento , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/tratamiento farmacológico , Hipersensibilidad a los Alimentos/epidemiología , Hipersensibilidad a los Alimentos/inmunología , Humanos , Inmunoglobulina E/inmunología , Masculino , Estudios Prospectivos , Calidad de Vida , Rinitis Alérgica/sangre , Rinitis Alérgica/tratamiento farmacológico , Rinitis Alérgica/epidemiología , Rinitis Alérgica/inmunología , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Proteolytic activities released by overnight incubation of Antwerpeen Trypanozoon antigenic type (AnTat) 1.1 trypanosomes at 4 degrees C in pH 5.5 and pH 7.0 phosphate/glucose buffers were analyzed in the supernatants obtained after centrifugation of the parasite suspensions. The assays used the fluorogenic substrates N-alpha-benzyloxycarbonyl-L-phenyl-alanyl-L-arginine-7-amido-4- methylcoumarin (Z-Phe-Arg-NMec) and N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine-7- amido-4-methylcoumarin (Z-Arg-Arg-NMec) at two different pHs (6.0 and 8.3). Z-Phe-Arg-NMec hydrolysis was inhibited by 2 microM L-trans-epoxysuccinyllencylamido(4-guanidino)butane (E-64) to a greater extent in the pH 7.0 supernatant than in the pH 5.5 supernatant. Z-Arg-Arg-NMec hydrolysis by the two supernatants was not significantly inhibited by 2 microM E-64. At pH 8.3 this activity was increased more than 2-fold by the addition of dithiothreitol. The hydrolysis activities were analyzed in collected eluates after fractionation of the supernatants by gel permeation high-performance liquid chromatography. Z-Phe-Arg-NMec hydrolytic activity inhibited by 2 microM E-64 was maximal at a retention time of 33 min (approx. Mr 30,000). In addition, a hydrolytic activity against the substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec gave a peak showing a maximum at a retention time of 29 min (approx. Mr 70,000).
Asunto(s)
Endopeptidasas/metabolismo , Trypanosoma brucei brucei/enzimología , Animales , Tampones (Química) , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Dipéptidos/metabolismo , Endopeptidasas/aislamiento & purificación , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Cinética , Especificidad por SustratoRESUMEN
The amino acid sequence of the proline-rich glycoprotein from human parotid saliva was studied using different preparations: native glycoprotein, tryptic glycopeptides, and their chemically deglycosylated homologous derivatives. The results indicate very similar structural features of the proline-rich glycoprotein and the tryptic glycopeptides and suggest that the peptide backbone of the glycoprotein comprises several repetitive domains containing one carbohydrate-peptide linkage and a proline-rich sequence. These data are in fair agreement with a preceding circular dichroism study (Aubert, J.P., Porchet, N., Boersma, A., Loucheux-Lefevbre, M.H. and Degand, P. (1982) Biochim. Biophys. Acta 704, 361-365).
Asunto(s)
Glicoproteínas/análisis , Glándula Parótida/metabolismo , Prolina/análisis , Saliva/análisis , Secuencia de Aminoácidos , Carbohidratos/análisis , Cromatografía en Gel , Humanos , Fragmentos de Péptidos/análisis , Tripsina/análisisRESUMEN
The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.
Asunto(s)
Endopeptidasas/aislamiento & purificación , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Ditiotreitol/farmacología , Endopeptidasas/química , Endopeptidasas/metabolismo , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Leupeptinas/farmacología , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Trypanosoma brucei brucei/metabolismoRESUMEN
The cross-reacting antigenic determinant in the variant surface glycoproteins (VSGs) of Trypanosoma equiperdum was studied by testing the ability of VSG glycopeptides to bind heterologous anti-VSG sera. VSG glycopeptide purification revealed the presence of 3 oligosaccharide sidechains on the mature VSG. These consist of two sidechains containing only mannose and glucosamine and a third containing galactose and mannose (in a 5:1 ratio) as well as phosphorous and ethanolamine. This phosphorylated fragment completely blocked the binding of VSG to heterologous anti-VSG and therefore contained the cross-reacting determinants.
Asunto(s)
Antígenos de Superficie/aislamiento & purificación , Epítopos/aislamiento & purificación , Glicopéptidos/aislamiento & purificación , Trypanosoma/inmunología , Animales , Fenómenos Químicos , Química , Reacciones Cruzadas , Glicopéptidos/inmunología , Fosforilación , Unión ProteicaRESUMEN
The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/metabolismo , Animales , Transporte Biológico Activo , Polaridad Celular , Células Epiteliales/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Humanos , Mucinas/química , Mucinas/metabolismo , Transducción de Señal , Red trans-Golgi/metabolismoRESUMEN
The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.
Asunto(s)
Neoplasias del Colon/metabolismo , Mucosa Gástrica/metabolismo , Microsomas/enzimología , Mucinas/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Animales , Glicosilación , Humanos , Mucina 5AC , Porcinos , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Salivarian trypanosomes have the ability to evade the immune response of their hosts by the sequential expression of different cell surface glycoproteins. Among the isolated specific antigens from cloned variants of Trypanosoma equiperdum, a structural study was undertaken on two immunologically cross-reacting variant surface glycoproteins, and results concerning the basic antigenic type are reported. The glycoprotein was cleaved by cyanogen bromide, and amino acids of several purified fractions obtained by gel filtration chromatography of this cleavage mixture were sequenced by automated Edman degradation. Sequencing in particular allowed the identification of the N-terminal portion of the molecule (residues 1-74). Sugar compositions of the fractions have demonstrated the presence of at least two carbohydrate moieties in the glycoprotein. Using a subsequent enzymatic subcleavage we were able to locate the first glycosylation site in position 57. An important observation was that the first oligosaccharide identified was rich in mannose and devoid of galactose.
Asunto(s)
Glicoproteínas/análisis , Trypanosoma/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Carbohidratos/análisis , Bromuro de Cianógeno , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Glicoproteínas Variantes de Superficie de TrypanosomaRESUMEN
Human bronchial secretions were examined for chemical components and rheological properties. Proline-rich polypeptides (PRP) obtained by ultrasonic treatment and by contact with a cationic resin (AG 50WX2) were purified by gel-filtration chromatography and high-voltage electrophoresis. The chemical composition of these components allowed a classification according to their proline, glycine, glutamic acid and lysine contents. Rheological experiments suggest a biological role for the PRP in the fibrillar structure of sputum.
Asunto(s)
Bronquios/metabolismo , Péptidos/análisis , Prolina/análisis , Aminoácidos/análisis , Resinas de Intercambio de Catión , Fenómenos Químicos , Química , Cromatografía en Gel , Electroforesis en Papel , Humanos , Concentración de Iones de Hidrógeno , Péptidos/aislamiento & purificación , Reología , Ultrasonido , ViscosidadRESUMEN
A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.
Asunto(s)
Oligopéptidos/aislamiento & purificación , Alcohol Polivinílico/química , Secuencia de Aminoácidos , Electroforesis Capilar , Glicosilación , Indicadores y Reactivos , Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
The acceptor specificity of three major isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyltranferases (murine recombinant proteins GaNTase-T1, -T2 and -T3) was investigated using the synthetic peptide (GTTPSPVPTTSTTSAP) containing clusters of threonine residues mimicking the mucin tandem repeat unit of MUC5AC. The O-glycosylated products obtained after in vitro reactions were fractionated by capillary electrophoresis and the purified glycopeptides were characterized by MALDI mass spectrometry (number of O-GalNAc residues) and by Edman degradation (site location). A maximum of three GalNAc residues was transferred into the MUC5AC motif peptide and the preferential order of incorporation for each GaNTase isoform was determined. Our results suggest that clusters of threonine appear to be essential for site recognition of peptide backbone by the ubiquitous GaNTases and also support the notion that the different GaNTase isoforms with varying substrate specificities are involved in a hierarchical order of O-glycosylation processing of the mucin-type O-glycoproteins.
Asunto(s)
Isoenzimas/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Péptidos/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Electroforesis Capilar , Glicosilación , Humanos , Ratones , Mucinas/química , Péptidos/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
The authors describe 68 articular fractures of the lower end of the radius in which a postero-medial accessory fragment was displaced. They have made a study of the anatomy and fibrous connections of the fragment and conclude that this fragment is best maintained reduced by a transverse pin passed from the ulna towards the radius. They attach great importance in such fractures to injury to the triangular radio-ulnar ligament and to inferior radio-ulnar dislocation.
Asunto(s)
Clavos Ortopédicos , Fijación Interna de Fracturas/métodos , Fracturas del Radio/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fracturas del Radio/patología , Fracturas del Radio/fisiopatologíaAsunto(s)
Glándula Parótida , Saliva/análisis , Acetilglucosamina/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Cromatografía en Gel , Fucosa/análisis , Galactosa/análisis , Glicopéptidos/análisis , Glicopéptidos/aislamiento & purificación , Glicoproteínas/análisis , Glicoproteínas/aislamiento & purificación , Humanos , Manosa/análisis , Papaína , Pronasa , Ácidos Siálicos/análisisAsunto(s)
Ácido Hialurónico/análisis , Mesotelioma/metabolismo , Derrame Pleural/análisis , Aminoácidos/análisis , Animales , Biopsia , Bovinos , Condroitín/aislamiento & purificación , Cromatografía , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis , Glicosaminoglicanos/aislamiento & purificación , Heparina/aislamiento & purificación , Humanos , Ácido Hialurónico/aislamiento & purificación , Hialuronoglucosaminidasa , Hidrólisis , Queratinas/aislamiento & purificación , Masculino , Mesotelioma/diagnóstico , Metástasis de la Neoplasia , Proteínas de Neoplasias/análisis , Oxidación-Reducción , Ácido Peryódico , Neoplasias Pleurales/diagnóstico , Proteínas/análisis , Coloración y Etiquetado , Ácidos Sulfúricos/aislamiento & purificación , Testículo/enzimología , Cuerpo Vítreo/análisisRESUMEN
A mucin-motif peptide in the one-letter code T T T P S P P M T T P I T P P A, representative of the human intestinal mucin tandem repeat sequence (MUC2), containing several threonine residues in clusters, was used as an acceptor substrate to investigate the effect of peptide structure on the activity of crude preparation of human gastric UDP-GalNAc:polypeptide N-acetyl galactosaminyltransferases. High-performance liquid chromatography was performed to separate the different products of the in vitro O-glycosylated reaction. The electrospray mass spectrometry was used to identify the different masses (m/z) of these products. Although the m/z of glycopeptide(s) could be higher than the detection limits of the spectrometer, an accurate study of the doubly charged ions allowed us to demonstrate the linkage of more than two sugars. Hence, the peptide MUC2 will accept at least four carbohydrate residues but the exact substituted positions should be confirmed by further sequence determination.