Uptake of dolichol into cultured cells.
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Animals↗
Biomedical subjects
T D Butters
Publications and source records attributed to T D Butters.
Isolation and characterization of mosquito cell membrane glycoproteins.
Plasma membranes have been purified from an established cell line, Mos 20A of Aedes aegypti, and analysed for glycoprotein and polypeptide constituents by isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis. A major glycoprotein of molecular weight 110 000 carrying binding sites for concanavalin A and soybean agglutinin has been purified to homogeneity. Although located on the cell surface, the 110 kdalton glycoprotein is not labelled by lactoperoxidase-catalysed radioactive iodination of whole cells. Analysis indicated the presence of N-glycans, containing on average nine mannose residues, and the N-acetylglucosaminyl-beta 1, 4-N-acetylglucosamine sequence. In addition, O-glycosidically linked N-acetylgalactosamine residues are present.
Aedes↗
Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin.
A cultured cell line of the mosquito, Aedes aegypti, is sensitive to tunicamycin as expected from the ability of crude membrane preparations to catalyse the formation of N-acetylglucosamine-linked dolichyl pyrophosphate. Formation of dolichylphosphomannose was also detected and this reaction was totally insensitive to tunicamycin. Incorporation of radioactive mannose into total acid-precipitable glycoproteins was inhibited greater than 90% in whole cells by tunicamycin, while the incorporation of leucine and glucosamine was less affected. Separation of the radioactive hexosamines from acid hydrolysates of cells incubated with [14C]glucosamine and tunicamycin showed predominant labelling of galactosamine, whereas in control cells not treated with the drug both glucosamine and galactosamine were labelled equally. Evidently, mosquito cells synthesise N-glycosidically linked carbohydrate chains assembled through tunicamycin-sensitive steps involving dolichyl pyrophospho-oligosaccharides, and O-glycosidically linked chains rich in N-acetylgalactosamine, the assembly of which is unaffected by tunicamycin. These results support structural evidence (Butters, T.D. and Hughes, R.C. (1981) Biochim. Biophys. Acta 640, 655-671) for the presence of high mannose N-glycans and N-acetylgalactosamine-rich O-glycans in mosquito cell glycoproteins. The absence of complex N-glycans was confirmed by the demonstration of negligible activities of N-acetylglucosaminyl-, galactosyl- and sialyltransferases responsible for assembly of the terminal sequences of N-glycans of mature mammalian glycoproteins.
Aedes↗
Cell surface molecules involved in fibronectin-mediated adhesion. A study using specific antisera.
Attachment and spreading of baby hamster kidney (BHK) fibroblasts to fibronectin-coated surfaces can be inhibited by antibodies, and the (Fab) 2 fragments, prepared against the cells. The antibodies reacted specifically with ricin-binding glycoproteins of the cell surface, the major components having molecular weights of 130-140 K. The antibodies reacted also with mouse fibroblasts (L-cells) and Chinese hamster ovary (CHO)-cells and inhibited their fibronectin-mediated adhesion to inert surfaces. Antisera raised against material isolated from the underside of BHK cells spread out on fibronectin-coated substrata using a cleavable, photolabile heterobifunctional cross-linking reagent (Aplin et al. Exptl. Cell Res. (1981) 134, 488-494) also affected the interactions of cells with fibronectin-coated surfaces. The antibodies stained the periphery of BHK cells and L-cells in a discontinuous periodic manner and immunoprecipitated as a major component a low molecular weight (approximately 47 K) glycoprotein from cell extracts. These results indicate that different specific antisera modify cell-substratum adhesion, probably by interacting with different cell surface components. The properties of multiple factors involved in the attachment and spreading of cells to suitably adhesive substrata are discussed.
Animals↗
Effects of tunicamycin on insect cells.
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Animals↗
Inhibition of fibronectin-mediated adhesion of hamster fibroblasts to substratum: effects of tunicamycin and some cell surface modifying reagents.
Using baby hamster kidney (BHK) fibroblasts we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the ability of trypsinized cells to attach and spread onto fibronectin. Tunicamycin inhibited mannose incorporation into total acid-precipitable glycoproteins by at least 95% while glucosamine and leucine incorporation were less or hardly inhibited. Hydrolysis and analysis of [3H]glucosamine-labelled glycoproteins showed that radioactivity incorporated into cells exposed to tunicamycin was present predominantly as galactosamine, presumably present in O-glycosidically linked glycan chains whose assembly is insensitive to the drug. Treated cells exhibit reduced amounts of surface-associated fibronectin and adhere relatively poorly to plastic or collagen surfaces pre-coated with plasma or BHK cell-derived fibronectins at the minimum concentrations required to induce nearly quantitative attachment and spreading of untreated cells. Drug-treated cells do adhere and spread into a bipolar configuration on surfaces saturated with fibronectin. Cells treated with tunicamycin and then grown in the absence of the drug revert to a more normal behaviour, indicating that under certain conditions the effects of the drug are reversible. Fibronectin-mediated spreading of trypsinized BHK cells is also inhibited by pre-treatment of cells with several non-penetrating reagents reactive with cell surface amino groups, namely pyridoxal phosphate, trinitrobenzene sulphonate and fluorescein 5-isothiocyanate. Analysis of surface substitution indicates a strong correlation between the extent of amino group substitution and inability of treated cells to interact with a fibronectin lattice. While the extent of attachment under these conditions is normal, cells pretreated with a specific non-penetrating thiol reagent, p-chloromercuribenzenesulphonate fail to attach to fibronectin-coated culture dishes in a dose-dependent fashion, indicating that a biochemical distinction can be made between the processes of attachment and spreading. We conclude that both N-glycosidically linked carbohydrate moieties of BHK cell surface glycoproteins and primary amine groups present in surface proteins or lipid head groups play a role in interactions of cells with fibronectin, leading to the formation and maintenance of a stable well-spread morphology. Both N-linked glycans and surface sulphydryl groups appear to be required for an attachment process which precedes spreading.
Amines↗
Liposome-mediated ricin toxicity in ricin-resistant cells.
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Animals↗
Lectin binding to mosquito Aedes aegyptii and human KB cells: structural comparisons of membrane oligosaccharides.
High capacity adsorbents for lectins, including Lotus tetragonolobus L-fucose-binding protein, were readily prepared by conjugation of monosaccharides with commercially available, epoxy-activated Sepharose. Purified, radioiodinated lectins were bound to cells of the mosquito Aedes aegyptii and of human KB tumour. Relative to human KB cells, mosquito cells bound less of lectins specific for the sugars (L-fucose and D-galactose) that are terminal residues in many mammalian glycoproteins, whereas the number of binding sites of lectins specific for core-region sugars (D-mannose and 2-acetamido-2-deoxy-D-glucose) were similar. Neuraminidase, which greatly enhanced binding of peanut agglutinin or soybean agglutinin to human KB cells, had negligible effects on binding of these lectins to mosquito cells. The comparative structures of surface oligosaccharides of mosquito and KB cells are discussed in relation to the lectin-binding studies.
Aedes↗
Interactions of KB-cell glycoproteins with an adenovirus capsid protein.
Glycoprotein material extracted from human KB cells with a flurocarbon, trichlorotrifluoroethane (Arklone P), into a waste-soluble fraction binds to fibre, a structural protein of the adenovirus type-5 capsid. The fibre-binding glycoprotein(s) were purified by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on a fibre-Sepharose support. The purification procedure also includes a trypsinization step which eliminates the bulk of contaminating KB cell proteins present in the aqueous fraction without appreciably affecting the activity of the fibre-binding glycoprotein(s). Some comparison is made of the membrane-bound receptors for adenovirus and the water-soluble fibre-binding glycoprotein(s).
Adenoviridae↗
Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins.
1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5'-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.
Autoradiography↗
Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes.
1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.
Adenoviridae↗