RESUMO
The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.
Assuntos
Nanopartículas de Magnetita , Animais , Bovinos , Glicoproteínas , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Peptídeos , PolissacarídeosRESUMO
The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/antagonistas & inibidores , Didelphis/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Polissacarídeos/análise , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Venenos de Crotalídeos/metabolismo , Glicosilação , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
An acid trehalase from Rhizopus microsporus var. rhizopodiformis was purified to apparent homogeneity. The molecular weight by SDS-PAGE (60 kDa) or Sephacryl S-200 filtration (105 kDa) suggested a homodimer. The carbohydrate content was 72%. Endoglycosidase H digestion resulted in one sharp band of 51.5 kDa in SDS-PAGE. pH and temperature optima were 4.5 and 45 degrees C, respectively. The isoelectric point was 6.69 and activation energy was 1.14 kcal mol(-1). The enzyme was stable for 1 h at 50 degrees C and decayed at 60 degrees C (t50 of 1.3 min.). Apparent KM for trealose was 0.2mM. Immunolocalisation studies showed the enzyme tightly packed at the surface of the cells.
Assuntos
Rhizopus/química , Rhizopus/enzimologia , Trealase/metabolismo , Carboidratos/análise , Dimerização , Ácido Edético/farmacologia , Ativadores de Enzimas/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Metais/farmacologia , Peso Molecular , Temperatura , Trealase/química , Trealase/isolamento & purificação , Trealose/metabolismoRESUMO
Growth hormone (GH) releasing hormone (GHRH) transgenic mice were used to examine the influence of GH on GH receptor (GHR) and membrane-associated GH binding protein (MA-GHBP) levels by means of specific radioimmunoassays and Western blot analysis, since MA-GHBP was described as the major constituent of somatogenic binding to liver membranes in mice. In transgenic animals, a 10-fold increment over normal values was found for hepatic somatogenic binding that could be accounted for by a 3--4-fold increase in GHR and a 9-fold augmentation of MA-GHBP levels. The apparent molecular weight of MA-GHBP was smaller than that of serum GHBP, a difference that was partially abolished by endoglycosidase F digestion. In vivo treatment of female mice with 17 beta-estradiol led to an unexpected down-regulation of MA-GHBP and GHR by 60--75% only in transgenic animals. MA-GHBP and GHR levels are strongly up-regulated by GH, although MA-GHBP up-regulation is much more important than that of GHR.
Assuntos
Proteínas de Transporte/metabolismo , Estradiol/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Camundongos Transgênicos , Receptores da Somatotropina/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Membrana Celular/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Radioimunoensaio , Regulação para CimaRESUMO
A new hypothesis for activation of the contact system of plasma proteolysis (i.e., the plasma kallikrein/kinin system) is presented. Kininogens have a multiprotein receptor on endothelial cells which consists of at least cytokeratin 1, urokinase plasminogen activator receptor, and gC1qR. When contact proteins (high molecular weight kininogen followed by prekallikrein) assemble on the kininogen receptor on endothelial cells, an endothelial cell membrane cysteine protease is expressed to activate prekallikrein to kallikrein. On endothelial cells, prekallikrein activation is independent of factor XIIa activation. Activation of prekallikrein on endothelial cells results in kallikrein cleaving its receptor high molecular weight kininogen to liberate bradykinin. Bradykinin liberation stimulates release of tissue-type plasminogen activator from endothelial cells. Kallikrein formation also results in kinetically favorable pro-urokinase activation on endothelial cells with subsequent plasminogen activation. In addition to stimulating cellular fibrinolysis, kininogens contribute to the constitutive anticoagulant nature of the intravascular compartment. Kininogens block calpain's participation in forming the heterodimeric complex of platelet integrin alpha IIb beta 3. Kininogens also block thrombin from binding to the thrombin receptor(s) on platelets. Last, kininogens prevent thrombin from cleaving protease activated receptor 1 after arginine41. These combined data indicate a biologic system for activation of the plasma kallikrein/kinin system and physiologic consequences as result of this activation.
Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Sistema Calicreína-Cinina , Cininogênios/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Plasma/metabolismo , Bradicinina/metabolismo , Endotélio/química , Fibrinólise , Pré-Calicreína/metabolismo , Receptores de Peptídeos , Trombina/antagonistas & inibidoresRESUMO
The anionic form of glutathione S-transferase from human (GST pi) and rat (GST Yp) sources has been shown to exist in multiple forms which have similar molecular weights but different isoelectric points (pIs). Treatment with endoglycosidase H caused the acidic forms of GST Yp to be converted to proteins with more basic pIs as compared to the untreated control mixtures, suggesting that an N-linked mannose moiety containing acidic residues had been removed. Inability to detect these carbohydrates by techniques requiring unsubstituted vicinal hydroxyls further suggested acidic substitutions on the sugar moiety. GST pi/Yp carbohydrate modifications were also identified by differential staining procedures. These data represent the first indication that glycosylation of GST can occur. Additionally, this may offer an explanation for the often seen microheterogeneity within a class of GST isozymes.
Assuntos
Glutationa Transferase/química , Isoenzimas/química , Animais , Metabolismo dos Carboidratos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Glicosilação , Humanos , Técnicas Imunoenzimáticas , Ponto Isoelétrico , Isoenzimas/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Ratos , Coloração e RotulagemRESUMO
High mannose-type, N-linked oligosaccharides devoid of glucose units may be glucosylated directly from UDP-Glc in mammalian, plant, fungal and protozoan cells. The glucosylated compounds thus formed (protein-linked Glc1Man5-9GlcNAc2, depending on the organisms) are immediately deglucosylated by glucosidase II, an enzyme located, the same as the glucosylating activity, in the endoplasmic reticulum. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated in the trypanosomatid Crithidia fasciculata (a microorganism transferring Man7GlcNAc2 in protein N-glycosylation) cells of the parasite were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and/or castanospermine that were several hundred-fold higher than those required to inhibit 50% of the activity of the protozoan enzyme. The inhibitors did not affect the cell growth rate and, although glucose analogs, did not interfere with the entry of glucose into the cells. About 40-43% of total N-linked oligosaccharides appeared to be glucosylated. As on the average there are several N-linked oligosaccharides per glycoprotein, more than 40-43% (but probably not all of them) are transiently glucosylated in the endoplasmic reticulum.
Assuntos
Crithidia/metabolismo , Glucose/metabolismo , Glicoproteínas/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , 1-Desoxinojirimicina , Acetilglucosaminidase/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Eletroforese em Papel , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Glucosidases/metabolismo , Glicosilação , Indolizinas/farmacologia , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Microssomos/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Trissacarídeos/metabolismoRESUMO
The structure of the N-linked oligosaccharide of the 85-kDa surface glycoprotein (Tc-85) from the infective trypomastigote form of Trypanosoma cruzi was investigated. Tc-85 metabolically labeled with [14C]glucose was purified by affinity chromatography on wheat germ agglutinin-Sepharose. Binding to the lectin was lost on treatment of Tc-85 with neuraminidase. The N-linked asialo-oligosaccharide was released by endo-beta-N-acetylglucosaminidase F digestion of asialo-Tc-85 and was further analyzed using specific exoglycosidases. [14C]fucose was detected after alpha-L-fucosidase treatment or mild acid hydrolysis. The afucosyl oligosaccharide was 3H-labeled by the galactose oxidase-NaB3H4 method. [3H]Galactose was released by alpha-galactosidase, and only then was beta-galactosidase effective in removing another galactose. The gal(alpha 1-3)gal unit was demonstrated by periodate oxidation studies on the [3H]galactose-labeled asialo-glycoprotein. The presence of gal(alpha 1-3)gal in Tc-85 could be related to the recent finding of elevated antibody levels against this epitope in patients with Chagas' disease.
Assuntos
Glicoproteínas/análise , Proteínas de Protozoários/análise , Trypanosoma cruzi/fisiologia , Acetilglucosaminidase , Animais , Dissacarídeos/análise , Fucose/análise , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Estrutura Molecular , Ácidos Siálicos/análiseRESUMO
The 90-kDa antigen, previously identified by the monoclonal antibody 1G7 to be a stage-specific surface protein of metacyclic trypomastigotes of Trypanosoma cruzi, has been further characterized in this study. Experiments of metabolic labeling with [35S]methionine, [2H]mannose and [3H]galactose revealed that the 90-kDa antigen is the main glycoprotein synthesized by metacyclic forms (G strain). Through pulse-chase experiments with [35S]methionine-labeled metacyclic trypomastigotes, it was found that the antigen is synthesized as a 75-kDa precursor polypeptide that is rapidly processed to the mature 90-kDa molecule. When metacyclic trypomastigotes were treated with tunicamycin, the production of 90-kDa antigen was greatly diminished, and the 75-kDa species, which was also expressed on the cell surface, accumulated. Concanavalin A bound strongly to the 90-kDa antigen, but failed to recognize the 75-kDa polypeptide. Treatment of neuraminidase had no effect on the 90-kDa antigen, whereas digestion by endoglycosidase H generated a polypeptide of 82 kDa. Altogether these data indicate that the 90-kDa antigen is a glycoprotein containing N-linked oligosaccharide side chains of the high-mannose type. The 90-kDa glycoprotein may be involved in the process of host cell invasion, since the internalization of metacyclic forms into Vero cells was partially inhibited by monoclonal antibody 1G7.
Assuntos
Antígenos de Protozoários/biossíntese , Antígenos de Superfície/biossíntese , Trypanosoma cruzi/imunologia , Acetilglucosaminidase , Animais , Anticorpos Monoclonais , Glicosilação , Radioisótopos do Iodo , Manose/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peso Molecular , Neuraminidase , Processamento de Proteína Pós-Traducional , Trypanosoma cruzi/patogenicidade , Células VeroRESUMO
An assay for UDP-Glc:glycoprotein glucosyltransferase was developed. Incubation of rat liver microsomes with UDP-[14C]Glc led to the formation of hot trichloroacetic acid insoluble material identified as protein-linked Glc1Man7-9GlcNAc2. Addition of 8 M urea-denatured thyroglobulin to the incubation mixtures stimulated up to 10-12-fold the formation of the same compounds but only in the presence of detergents. Native thyroglobulin was ineffective. Several experiments indicated that the stimulation was due to the transfer of glucose residues from UDP-Glc to high-mannose oligosaccharides in urea-denatured thyroglobulin and that this transfer reaction did not involve dolichol mono- or diphosphate derivatives as intermediates. The glycoprotein glucosylating activity was mainly located in the endoplasmic reticulum and could glucosylate glycopeptides derived from the digestion of thyroglobulin with an unspecific protease. Glucosylation of oligosaccharides in those glycopeptides occurred, however, at a rate at least 2 orders of magnitude slower than that of the same compounds in urea-denatured thyroglobulin. Tryptic digestion of urea-denatured thyroglobulin did not affect its glucosylation rate. The structure of Glc1Man9GlcNAc2 linked to urea-denatured thyroglobulin was identical with that of Glc1Man9GlcNAc2-P-P-dolichol. The assay of UDP-Glc:glycoprotein glucosyltransferase allowed detection of the activity in microsomal membranes in which endogenous acceptors appeared to be absent or almost absent, such as those derived from mung bean, Mucor rouxii, Crithidia fasciculata, and Trypanosoma cruzi cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicoproteínas/metabolismo , Hexosiltransferases , Proteínas de Membrana , Microssomos/metabolismo , Plantas/metabolismo , Streptomyces griseus/metabolismo , Trypanosoma cruzi/metabolismo , Acetilglucosaminidase/metabolismo , Animais , Crithidia/metabolismo , Glucose/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Masculino , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Microssomos Hepáticos/metabolismo , Mucor/metabolismo , Peptídeo Hidrolases/metabolismo , Desnaturação Proteica , Ratos , Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/metabolismo , Transferases/metabolismoRESUMO
Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.
Assuntos
Anticorpos Antiprotozoários/imunologia , Epitopos/análise , Galactose/análise , Glicoproteínas de Membrana/análise , Trypanosoma cruzi/imunologia , Acetilglucosaminidase , Animais , Antígenos de Protozoários/análise , Antígenos de Superfície/análise , Sítios de Ligação de Anticorpos , Western Blotting , Linhagem Celular , Soros Imunes/análise , Rim , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Glicoproteínas de Membrana/isolamento & purificação , Peptidoglicano/isolamento & purificação , Fosfolipídeos/isolamento & purificação , Testes de Precipitina , Radioimunoensaio , Trypanosoma cruzi/análiseRESUMO
As previously reported, incubation of liver dolichol-P, UDP-[14C]Gal, and a particulate preparation of Acetobacter xylinum leads to the synthesis of dolichol-P-[14C]Gal (P. Romero, R. Garcia, and M. Dankert (1977) Mol. Cell. Biochem. 16, 205-212). It is now reported that upon incubation of the latter with rat liver microsomes, [14C-galactose]-Gal1Man9GlcNAc2-P-P-dolichol and [14C-galactose]Gal1Glc1Man9GlcNAc2-P-P-dolichol are formed. The galactosyl residues appeared to be (1,3)-linked in the same positions as the glucose units in the respective physiological compounds. No lipid-linked Gal1Glc2Man9GlcNAc2 was formed, thus strongly suggesting the presence of at least two dolichol-P-Glc:dolichol-P-P-oligosaccharide glucosyltransferases, only one of which is able to use dolichol-P-Gal as substrate. Incubation of the galactosylated dolichol-P-P derivatives with rat liver microsomes led to the transfer of the oligosaccharides to microsomal proteins. No endogenous, membrane-bound glycosidases were able to remove the galactose residues but mannose units were excised by endogenous neutral mannosidases.