Purification and characterization of the mouse thymocyte Thy-1 glycoprotein.
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ABSTRACT:
SUBMITTER: Carlsson SR
PROVIDER: S-EPMC1154409 | biostudies-other | 1983 Jun
REPOSITORIES: biostudies-other
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Project description:Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary 'complex-type' with terminal fucose; IIA, biantennary 'complex-type' without fucose; IIB, biantennary 'complex-type' with fucose; III, a mixture of 'high-mannose' chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of 'high-mannose' type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with 'complex-type' chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three 'complex-type' chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.
| S-EPMC1144049 | biostudies-other
Project description:We have purified from chicken forebrain a membrane glycoprotein that is enriched in purified synaptic membranes and has an apparent mol.wt. of 22 800 in 15% sodium dodecyl sulphate/polyacrylamide gels. This molecule was compared with rat and human brain Thy-1 glycoproteins purified by the same procedure in order to determine whether it could be a homologue of Thy-1. Although polyvalent heterologous antisera raised against the rat and chicken molecules showed no immunological cross-reactivity with the other glycoprotein, a great deal of physical and chemical similarity was demonstrated between the chicken glycoprotein and rat Thy-1. Their apparent molecular weights, subcellular localization and amino acid and amino sugar compositions are very similar. C.d. spectra show that both molecules contain predominantly a beta-sheet and structure with no detectable alpha-helix. Electrophoretic analysis of the CNBr-cleaved molecules under reducing and non-reducing conditions shows that both molecules contain intramolecular disulphide bridges. Taken together these results suggest that the chicken brain glycoprotein is an immunologically distinct homologue of the mammalian Thy-1 glycoproteins.
| S-EPMC1152101 | biostudies-other
Project description:The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.
| S-EPMC1172418 | biostudies-other
Project description:PurposeThe goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones.Materials and methodsFor hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953-7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection.ConclusionThis study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.
| S-EPMC6082671 | biostudies-literature
Project description:Modified DNA aptamers incorporated with amino-acid like side chains or drug-like ligands can offer unique advantages and enhance specificity as affinity ligands. Thy-1 membrane glycoprotein (THY1 or CD90) was previously identified as a biomarker candidate of neovasculature in pancreatic ductal adenocarcinoma (PDAC). The current study developed and evaluated modified DNA X-aptamers targeting THY1 in PDAC. The expression and glycosylation of THY1 in PDAC tumor tissues were assessed using immunohistochemistry and quantitative proteomics. Bead-based X-aptamer library that contains 108 different sequences was used to screen for high affinity THY1 X-aptamers. The sequences of the X-aptamers were analyzed with the next-generation sequencing. The affinities of the selected X-aptamers to THY1 were quantitatively evaluated with flow cytometry. Three high affinity THY1 X-aptamers, including XA-B217, XA-B216 and XA-A9, were selected after library screening and affinity binding evaluation. These three X-aptamers demonstrated a high binding affinity and specificity to THY1 protein and the THY1 expressing cell lines, using THY1 antibody as a comparison. The development of these X-aptamers provides highly specific and non-immunogenic affinity ligands for THY1 binding in the context of biomarker development and clinical applications. They could be further exploited to assist molecular imaging of PDAC targeting THY1.
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Project description:The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.
| S-EPMC9130087 | biostudies-literature
Project description:The attachment of glycolipid anchors to the Thy-1 glycoprotein during biosynthesis was followed by the change of detergent-binding properties of biosynthetically labelled Thy-1 precursors upon phospholipase C treatment in the murine thymoma lines BW5147 and S1A. In S1A, 80% of the Thy-1 molecules were phospholipase-C-sensitive after a 2 min pulse with [35S]methionine, indicating that these molecules were already anchored via a glycolipid tail. In BW5147, 47% of the Thy-1 molecules had phospholipase-C-sensitive anchors attached after a 1.5 min labelling and, with longer pulses, this percentage rose to 76%. Tunicamycin did not block the addition of glycolipid anchors, and glycolipid attachment also occurred at 21 degrees C. The findings suggest that the attachment of glycolipid anchors occurs in the rough endoplasmic reticulum.
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Project description:Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app=(5.2±0.20)×10(2)M(-1)s(-1), and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×10(5)M(-1)s(-1). Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.
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Project description:The present paper describes the characterization of the Thy-1 molecule from rat brain. The molecule was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate. In the solubilized form the Thy-1 antigens were associated with a homogenous component with the following hydrodynamic properties: s20,w=2.2s,v=0.72ml/g and Stokes radius=3.0nm. The mol.wt. of the deoxycholateantigen complex was estimated to be 27000; these values are not significantly different from those obtained thymocyte Thy-1. Brain Thy-1 was further purified by affinity chromatography with lentil lectin coupled to Sepharose 4B, and more than 80% of the antigen was bound. The material eluted with methyl alpha-D-glucopyranoside was then filtered on a column of Sephadex G-200, and only one glycoprotein was found in the antigenically active fraction. On sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the glycoprotein was very similar to the Thy-1 from thymocytes that binds to lentil lectin. Its apparent mol.wt. on 12.5% acrylamide gels was 24000, and it electrophoresed as a symmetrical band. Brain Thy-1 was antigenically indistinguishable from thymocyte Thy-1 when analysed with rabbit antisera raised against brain or thymocyte Thy-1.
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Project description:Histidine-Proline-rich Glycoprotein (HPRG) is a plasma protein of vertebrates and several marine bivalves. Due to its multidomain structure consisting of several regions HPRG can interact with a variety of ligands, however the exact physiological role has not been discovered yet. Past purification approaches out of plasma or serum often led to co-purification of other proteins so that for a profound understanding of the function it is important to obtain a protein of high purity. Recent purification strategies were based upon metale chelate affinity chromatography followed by anion exchange chromatography or size exclusion chromatography, respectively. A large amount of serum albumin, the major plasma protein, also elutes from metale chelate affinity chromatography columns. Separation of rabbit HPRG from rabbit serum albumin could not be achieved via the above named methods by us. We present a method of purification of rabbit serum HPRG by means of metal affinity chromatography and preparative gel electrophoresis, which makes it possible to obtain HPRG practically devoid of impurities as assessed by mass spectrometry analysis. Moreover, we characterize the amount of glycosylation of HPRG and-to the best of our knowledge for the first time-the glycosylation pattern of rabbit HPRG.
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