Tritiated Galactose Research Articles

Suppression of beta 1,3galactosyltransferase beta 3Gal-T5 in cancer cells reduces sialyl-Lewis a and enhances poly N-acetyllactosamines and sialyl-Lewis x on O-glycans.

We investigated the role of beta 3 Gal-T5, a member of the beta 1,3galactosyltransferase (beta 1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of beta 3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both beta 1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-beta-galactosidase mostly releases NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal and NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAc beta 1-3Gal and type 2 chain oligosaccharides, including NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-alpha-GalNAc. These data indicate that beta 3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, beta 3Gal-T5 transcript, beta 1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.

Ethanol-induced increase of sphingosine recycling for ganglioside biosynthesis: a study performed on cerebellar granule cells in culture.

The effect of ethanol on ganglioside metabolism was assessed in cultured rat cerebellar granule cells. Cells were incubated in the presence of tritiated serine or galactose, and the synthesis of radioactive gangliosides was followed. The rate of de novo biosynthesis of gangliosides labeled in the oligosaccharide moiety (deriving from tritiated galactose) was not affected by the presence of ethanol. On the contrary, the biosynthesis of gangliosides labeled in the ceramide long chain base moiety (deriving from tritiated serine), dramatically decreased in the presence of alcohol. These results suggest that the gap between the extent of the biosynthesis of lipid and polar portions observed in the presence of ethanol, is filled by an increased recycling of sphingosine produced from ganglioside degradation. This hypothesis was confirmed by pulse-chase experiments with GM1 ganglioside, tritiated in the sphingosine moiety, and following radiolabeled gangliosides deriving from its metabolic processing. In fact, the radioactivity carried by gangliosides whose labeling could derive exclusively (GD1b + GT1b) or partially (GD1a) from the recycling of catabolic radiolabeled sphingosine, dramatically increased in ethanol-treated cells during the chase period. Taken together, these results suggest that ethanol increases ceramide sphingosine recycling for ganglioside biosynthesis.

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells.

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

Effect of Tunicamycin on the Uptake and Incorporation of Galactose in Hymenolepis diminuta

The effects of tunicamycin (TM) on the uptake and incorporation of tritiated galactose into the tegumental membrane and carcass from adult Hymenolepis diminuta were examined to assess the potential usefulness of this inhibitor for studying the function of the tapeworm surface glycocalyx. Hymenolepis diminuta adults (11 days old) were preincubated for 1 hr, pulsed for 30 min with [3H]galactose and [14C]leucine, and chased for 2 hr; replicate experiments were conducted in which all media contained no TM or TM at 10 micrograms/ml. Tunicamycin significantly inhibited the incorporation of tritiated galactose into the tapeworm's carcass and 30,000-g tegumental membrane fraction. Incorporation of tritiated galactose into the tapeworm's tegumental surface membrane also was inhibited significantly when expressed relative to the incorporation of [14C]leucine. Tunicamycin did not affect the amounts of free, i.e., soluble, [3H]galactose or [14C]leucine recovered from the tapeworms not did it affect the short-term (2 min) uptake of [3H]galactose by tapeworms. Thus, the inhibitory effect of TM appears to be at the level of protein glycosylation rather than carbohydrate (galactose) transport. The data indicate that TM might be useful for producing tapeworm surface membranes with diminished carbohydrate moieties.

Expression of Lectin-Binding Surface Glycoproteins during the Development of Schistosoma mansoni Schistosomula

Tegumental glycoproteins of Schistosoma mansoni cercariae, mechanically produced 24-hr and 48-hr schistosomula, and adult worms were radioiodinated with the Bolton-Hunter reagent, then isolated by lectin affinity chromatography. SDS-PAGE revealed Con A binding glycoproteins with apparent molecular weights of 180,000, 150,000, 43,000, and 30,000 in detergent extracts of the tegument of cercariae. These glycoproteins are retained by 24-hr mechanically produced, cultured schistosomula and are accompanied by the appearance of 2 additional labeled glycoproteins, mol. wt. 66,000 and 57,000. In 48-hr schistosomula, there is a marked increase in the relative size of the 66,000 mol. wt. peak. In contrast, the 57,000 mol. wt. glycoprotein is the major radiolabeled Con A binding component of the adult tegument; the other peaks are either reduced or absent in adults. Similar findings were obtained following affinity chromatography using immobilized Lens culinaris lectin or Ricinus communis agglutinin, and following metabolic labeling of glycoproteins with tritiated galactose.

Incorporation of tritiated galactose into galactocerebroside by cultured rat oligodendrocytes: effects of cyclic adenosine 3',5'-monophosphate analogues.

Cells dissociated from the forebrains of 21-day-old rats were enriched in oligodendroglia by Percoll gradient centrifugation, seeded on polylysine-coated surfaces, and cultured in a serum-containing medium. Incorporation by the cultures of tritium from D-[3H]galactose into the galactosyl residue of galactocerebroside (galC) increased in an almost linear fashion for 48 h with 1-8 muCi of D-[3H]galactose (30 mCi/mumol) per milliliter medium. Treatment for 2 days (day 1-3 after seeding) with 10(-4) M or 10(-3) M dibutyryl cyclic adenosine 3',5'-monophosphate (db cyclic AMP) or 10(-4) M 8-bromo cyclic AMP stimulated galC radiolabelling. Incorporation of D-[3H]galactose into galC during a terminal 48-h radiolabelling period was not stimulated when the cells were continuously treated with these cyclic AMP analogues for 8 rather than 2 days.

Modification of simian virus 40 large tumor antigen by glycosylation

The SV40-encoded transforming protein, large tumor antigen (T-ag), is multifunctional. Chemical modifications of the T-ag polypeptide may be important for its multifunctional capacity. T-ag is additionally modified by glycosylation. T-ag was metabolically labeled in SV40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. The identity of glycosylated T-ag was established by immunoprecipitation with a variety of T-ag-specific antisera, including monoclonal antibodies. Incorporation of labeled sugar into T-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. Labeled monosaccharides could be preferentially removed from T-ag with a mixture of glycosidic enzymes. In addition, galactose was removed from purified T-ag by acid hydrolysis and identified as such by thin-layer chromatography. T-ag oligosaccharides were resistant to treatment with EndoH, and glycosylation was not inhibited by tunicamycin. Together, these data strongly suggest that T-ag is glycosylated. Several characteristics, including lack of mannose labeling, EndoH resistance, and tunicamycin resistance, suggest that T-ag is not an N-linked glycoprotein. Rather, these properties are more consistent with the identification of T-ag as an O-linked glycoprotein.

Isolation and characterization of flat cells, a subpopulation of the embryonic chick retina

When the embryonic neutral retina is dissociated into single cells which are maintained in stationary culture, the neuronal cells associate on the surfaces of a second population which we refer to as flat cells. The flat cells appear in the culture in significant numbers after 2 days and are required for neuronal cell attachment. We have been able to isolate pure flat cells from early cultures of mixed retina cells and have identified several antigens which support the concept that these cells are related to the glia. The cells have been tested by immunofluorescence for glial fibrillary acidic protein and have been found positive. Cell surfaces were labeled by transfer of tritiated galactose from UDP-galactose to endogenous acceptors in the presence of exogenous galactosyl transferase. After SDS-PAGE and fluorography, the surface glycoproteins of flat cells were seen to be significantly different from those of the original retina, and from chick fibroblasts. Immunoelectron microscope studies of detergent-extracted flat cells have demonstrated a complex network of intermediate filaments and actin fibers. We conclude that the flat cells are derived from the glia subpopulation of the retina and have adapted to the tissue culture environment by assuming this configuration. The unique surface properties of flat cells may be related to their role as an intermediate substrate between the neuronal cells and the tissue culture dish.

Effect of carbenoxolone on the synthesis of glycoproteins and DNA in rat gastric epithelial cells.

The influence of carbenoxolone on the synthesis of glycoproteins in the surface mucous cells and the production of new cells in the rat gastric mucosa was studied by means of a vascular perfusion system. The rate of incorporation of tritiated galactose, glucosamine, serine, and sulphate in surface mucous cells, studied by autoradiography, was not affected by the addition of carbenoxolone to the drinking water. The sugar composition (determined by gas-liquid chromatography) of the gastric glycoproteins (isolated by centrifugation in CsCl), was not changed in carbenoxolone-treated rats. Compared with untreated animals, the number of [3H]-thymidine labelled nuclei per fundic pit increased by 38% to 76% in carbenoxolone-treated rats, implying a higher number of mitotically active cells. This results in an increased supply of young mucous cells; if this also proves to be true in human gastric mucosa, it may be relevant to the therapeutic effect of carbenoxolone.

Quantitative electron microscopic autoradiography of in vivo incorporation of 3H-choline, 3H-leucine, 3H-acetate and 3H-galactose in non-ciliated bronchiolar (Clara) cells of mice.

AbstractThe participation of Clara cells in the formation of alveolar surfactant and their general metabolism were studied in mice by quantitative autoradiography under electron microscopy after in vivo incorporation of four precursors: 3H‐choline, 3H‐leucine, 3H‐acetate and 3H‐galactose.Tritiated choline, a specific precursor of the dipalmitoyl‐phosphatidylcholine‐active fraction of the surfactant, is not incorporated into Clara cells. In contrast, 3H‐acetate is first incorporated actively into the endoplasmic reticulum and Golgi apparatus of these cells and, subsequently, in its secretory granules. It is suggested that acetate follows the general metabolism of the cell and/or is transformed into cholesterol. Tritiated leucine is incorporated into the secretory granules, although part of it remains more permanently in the cells. Tritiated galactose is actively incorporated, passes through the Golgi apparatus and eventually appears in the secretory granules.These results suggest that the Clara cell does not synthetize the tensio‐active fraction of the lining layer, as does pneumocyte II, but participates in the synthesis and secretion of proteins, glucides and possibly the cholesterol belonging to the hypophase.