Ganglioside labeled

Gangliosides labeled by intracranial injection of l-t(C]ManNAc were extracted from 1 g of brain, applied with solvent 1 (vertically) and solvent 2 (horizontally). Assayed fractions indicated by numbers (and ganglioside abbreviations) correspond to the PFC assay results in Figure 4. Fraction 6 refers to the area surrounding all the assayed spots (15). 

Understanding of ganglioside sialic acid biosynthesis has been aided by the use of radioactive ManNAc. This approach has been used to prepare whole rat-brain gangliosides labelled only in the sialic acid moiety (Quarles and Brady 1971, ROsner et al. 1973, Caputto et al. 1976, Yohe and Rosenberg 1977). Studies with radioactive Glc and GlcN suggested that the site of biosynthesis was in the... 

Kolodny, E. H., Brady, R. O., Quirk, J., and Kanfer, J. N., 1970, Preparation of radioactive Tay-Sachs ganglioside labeled in the sialic acid moiety, J. Lipid Res. 11 144. 

Caveolae can mediate the delivery of CtxB that binds to GM1 ganglioside at the plasma membrane and is delivered to intracellular compartments. Cholera toxin, produced by Vibrio cholerae, consists of five identical subunits B and one A chain. In addition to labeled SV40 and caveolin-1-GFP, CtxB is one of the most commonly used caveolae markers. However, two groups reported that the toxin is internalized by either a clathrin-independent caveolae pathway or a clathrin-dependent uptake, bringing its selectivity/specificity into question (31,81,118). We controlled the suitability of this marker for COS-7 cells pretreated with CPZ, mpCD, and filipin and as expected, the uptake was not influenced by CPZ treatment but was strongly decreased by the latter two (data not shown). 

In contrast to this work on whole brain, Kemp and Stoolmiller138 used cultured mouse-neuroblastoma cells for their study of incorporation of [ lH]2-acetamido-2-deox> -D-mannose into the sialic acid moiety of gangliosides. They were able to demonstrate clearly the precursor-product relationship among CMS, GM2, and GM1. They found that cultured NB41A cells incorporated [3H]2-acetamido-2-deoxy-D-man-nose into the sialic acid moiety of GM3 in less than 10 minutes. Labeled GM2 was not detected in cells incubated for less than 30 minutes, and measurable activity did not appear in G I1 until alter 60 to 90 minutes. These results further supported the concept that the pathway of synthesis of ganglio-tvpe gangliosides proceeds by way of GM3 —> GM2 — GM1. 

Thin Layer Chromotography. Unless otherwise stated, all thin layer chromotography was on plates coated with silica Gel G (E. Merck, Dramstadt). All the solvents were mixed on a volume basis. Neutral glycolipid fractions were developed in chloroform methanol water (60 35 6.5). Labelled fucolipid fractions were developed in chloroform methanol water (40 40 10). For separation of gangliosides, chloroform methanol 2.5N aqueous NH4OH (60 40 9) was used. 

Labelled Neutral Glycolipids. Neutral glycolipids were labelled with -galactose. As was seen with the ganglioside, the butyrate affected the neutral glycolipid patterns but the most marked alterations appeared to be due to changes in the lipid moeities. 

Figure 4. Thin-layer chromatographic fluorogrants of labeled gangliosides...

When cells were cultured in labelled fucose or galactose in the presence or absence of butyrate, alterations in the labelled glycolipids were observed. Treatment of all of the cell lines with butyrate did not markedly affect the incorporation of l hJ galactose in ganglioside per milligram of cell protein. 

Gangliosides were labelled with galactose. In the fetal cell lines (FHS) and SKCO-1 there was no marked difference between treated and untreated cells. In HT-29, SW-480, and Sw-620 cell lines, the amounts of Gm3 appeared to remain the same, but the distribution of Gm3 components was affected by butyrate. These changes, might be due to alterations in the lipid moieties or, alternatively, there might be an acetylation Of a hydroxyl group in the carbohydrate moiety, since it has been shown that the butyrate increases the amount of acetylated histones. 

Exposure of HeLa cells to butyrate had no effect on the activity of GM3-sialidase when GM3 specifically labeled in the sialic acid residue was used as substrate (Fig. 3a). We were unable to detect any "ecto"-sialidase activity in either control or butyrate-treated cells (14) although others have postulated that such an enzyme is important in regulating plasma membrane gangliosides (15,16). In contrast, the activity of the specific sialyl transferase involved in GM3 biosynthesis increased over 20-fold following butyrate treatment (Fig. 3b). The effect was specific as activities of the other glycosphingolipid transferases that could be measured in HeLa cells were not altered in butyrate-treated cells (4,8,17). 

N4TG1 cells were labelled with L H. GLcN and cultured in the presence of different drugs for 24 hrs. Gangliosides were isolated as described in the text. 

Exogenous GM1 ganglioside can be incorporated into the cell membrane and then act as a functional receptor. This was first shown by Cuatrecasas (11) who observed an increased binding capacity and lipolytic responsiveness of fat cells which had been soaked in GM1. Using tritium-labelled GM1-ganglioside, Holmgren... 

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