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T Pacuszka

Publications and source records attributed to T Pacuszka.

14 recordsLinked to original sources
PubMed1992

Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.

We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.

Animals↗
PubMed1992

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.

Affinity Labels↗
PubMed1991

Metabolism of cholesterol, phosphatidylethanolamine and stearylamine analogues of GM1 ganglioside by rat glioma C6 cells.

Tritium-labeled neoglycolipids consisting of the oligosaccharide of ganglioside GM1 attached to cholesterol (GM1OSNH-X-CHOL), phosphatidylethanolamine (GM1OS-PE) and stearylamine (GM1OSNHC18) were synthesized and their uptake and metabolism by GM1-deficient rat glioma C6 cells were determined. When the neoglycolipids were added to serum-free culture medium, all three were rapidly taken up by the cells and initially inserted into the plasma membrane based on their resistance to trypsin and their ability to bind cholera toxin. With time, the neoglycolipids underwent internalization as the ratio of cell-associated radioactivity to cell surface toxin binding increased; this process was slow for GM1OSNH-X-CHOL and GM1OS-PE and rapid for GM1OSNHC18. Analysis of lipids extracted from the cells indicated that the neoglycolipids also underwent metabolism to GD1aOS-based analogues. In addition, GM1OSNH-X-CHOL and GM1OSNHC18 were degraded to their GM2OS-based analogues, whereas GM2OS-PE was not detected. In contrast, large amounts of 3H were recovered in the medium from cells treated with GM1OS-PE and the label was associated with material that behaved neither as an oligosaccharide or a neoglycolipid. In the presence of monensin or chloroquine, metabolism of the three neoglycolipids was inhibited. Thus, GM1OS-based neoglycolipids were taken up by the cells, internalized and sorted both to the Golgi apparatus (sialylated to GD1aOS-based analogues) and to lysosomes (hydrolyzed to GM2OS-based analogues). The rate and extent of these processes, however, were strongly influenced by the nature of lipid moiety.

Amines↗
PubMed1991

Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1979

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland.

Bovine thyroid glands are known to contain a complex array of gandliosides. One of the predominant gangliosides was isolated analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as: (Formula: see text).

Animals↗
PubMed1978

Interaction of human chorionic gonadotropin with membrane components of rat testes.

Previous studies demonstrating that gangliosides interacted with thyrotropin and human chorionic gonadotropin (hCG) suggested that gangliosides participate in the transduction of the hormonal message across the target cell membrane. As a continuation of these investigations, we examined the effects of down-regulation of hCG receptors on the interaction of hCG with rat testis membrane components. Rat testes contained a complex ganglioside pattern that did not appear to change qualitatively or quantitatively after the injection of hCG into the animals, although testis membranes from hCG-treated rats lost their capacity to bind (125)I-labeled hCG ((125)I-hCG). Gangliosides extracted from the testes of control and treated animals were equally effective inhibitors of (125)I-hCG binding to testis membranes. However, inhibition of binding was observed only under conditions (pH 6.0, low ionic strength) such that unlabeled hCG (>2500-fold excess) did not block (125)I-hCG binding, and (125)I-hCG bound similarly to testis membranes from control and treated rats. Under conditions such that hCG binding was specific (blocked by 250-fold excess of unlabeled hCG), testis gangliosides were noninhibitory. Liposomes containing gangliosides from the testes of control or hCG-treated rats bound similar small amounts of (125)I-hCG. These same liposomes bound 50 and 1000 times more thyrotropin and cholera toxin, respectively, than hCG. Oligosaccharides derived from gangliosides did not inhibit (125)I-hCG binding to testis membranes nor did they alter the fluorescence of hCG conjugated with fluorescent probes, whereas the gangliosides themselves were inhibitory and enhanced the fluorescence intensity of the hCG derivatives. Exposure of testis membranes from hCG-treated rats to 4 M MgCl(2), which displaces bound hCG [Chen, Y.-D. I. & Payne, A. H. (1977) Biochem. Biophys. Res. Commun. 74, 1589-1596], did not restore their ability to bind (125)I-hCG. When membranes were solubilized with Triton X-100, a solubilized receptor was detected from testis membranes of control but not hCG-treated rats. These findings and the absence of demonstrable changes in the composition or quantity of rat testis gangliosides when hCG receptors are down-regulated suggest that gangliosides do not represent the primary binding determinants of hCG receptors.

Animals↗
PubMed1978

Enzymatic synthesis of H-active pentaglycosylceramide by human bone marrow fucosyltransferase.

Synthesis of two fucoglycolipides I and II was obtained from GDP-fucose and lacto-N-neotetraosylceramide using either solubilized or particulate enzymic preparation from human bone marrow. Fucoglycolipid I on the basis of its chromatographic mobility and immunoprecipitation with Ulex europeus lectin was identified as H blood group active penta glycosylceramide. Fucoglycolipid II did not react with Ulex europeus and its fucosyl residue was hydrolyzed by alpha (1 leads to 3/4)-L-fucosidase of Trichomonas fetus. Most probably the fucose residue of glycolipid II was linked to N-acetyloglucosamine by 1 leads to 3 linkage.

ABO Blood-Group System↗
PubMed1976

Enzymatic synthesis of two fucose-containing glycolipids with fucosyltransferases of human serum.

Lacto-N-neotetraosylceramide incubated with human serum fucosyltransferase preparations gave rise to two fucoglycolipids. The faster migrating fucoglycolipid I on the basis of its thin-layer chromatographic mobility, susceptibility to alpha(1 leads to 2) fucosidase from Trichomonas foetus, radio-immunoprecipitation with Ulex europeus lectin and studies with Oh (Bombay) sera was identified as H-active glycolipid (H-I). The most probable structure of fucoglycolipid II should be that with fucose linked alpha(1 leads to 3) to N-acetylglucosamine. Lactosylceramide, ceramide trihexoside and globoside were not substrates for human serum fucosyltransferases. Lacto-N-neotetraosyl ceramide served as a fucose acceptor for all serum preparations tested while asialoganglioside was a substrate only when serum preparations containing H-gene dependent alpha-2-L-fucosyltransferase were used. With asialoganglioside only one radioactive reaction product was formed.

Blood Group Antigens↗