Manzi A E, Sjoberg E R, Diaz S, Varki A
Department of Medicine, U.C.S.D. Cancer Center.
J Biol Chem. 1990 Aug 5;265(22):13091-103.
We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.
我们和其他人之前描述了黑色素瘤相关的癌胚糖鞘脂抗原9-O-乙酰-GD3，一种在外层唾液酸残基的9位被O-乙酰化的双唾液酸神经节苷脂。我们现在已经开发出方法来检测培养的黑色素瘤细胞以及来自这些细胞的富含高尔基体的囊泡中双唾液酸神经节苷脂的生物合成和周转情况。O-乙酰化选择性地表达于双唾液酸和三唾液酸神经节苷脂上，但不表达于单唾液酸神经节苷脂上，也不表达于糖蛋白结合的唾液酸上。用[3H]乙酸盐和[14C]葡糖胺对细胞进行双重标记，可将易于检测的标记引入神经节苷脂分子的每个组分中。对这种双重标记分子的脉冲追踪研究表明，O-乙酰基团的周转速度比母体分子快。当将来自这些细胞的富含高尔基体的囊泡与[乙酰-3H]乙酰辅酶A一起孵育时，主要的标记产物是双唾液酸神经节苷脂。在7位和9位均发现了[乙酰-3H]O-乙酰基团，这表明7-O-乙酰-GD3和9-O-乙酰-GD3都是由O-乙酰转移酶对内源性GD3作用而合成的。对代谢标记分子的分析证实了完整细胞中存在7-和9-O-乙酰化的GD3。令人惊讶的是，体外标记反应的主要3H标记产物不是O-乙酰-GD3，而是GD3，标记仅存在于唾液酸残基中。通过酶促和化学方法对标记的唾液酸进行片段化分析表明，3H标记仅存在于[3H]N-乙酰基团中。对来自完整细胞的双重标记唾液酸的分析还表明，来自[3H]乙酸盐的3H标记仅以[3H]N-乙酰基团的形式存在，而14C标记位于4位。对3H/14C比值的脉冲追踪分析表明，GD3和单唾液酸神经节苷脂GM3的N-乙酰基团的周转速度都比母体分子快。选择性高碘酸盐氧化表明，GD3的内层和外层唾液酸残基在体外反应中均掺入了3H标记，并且在脉冲追踪研究中显示出类似的N-乙酰化周转情况。综上所述，这些结果表明神经节苷脂唾液酸残基的O-和N-乙酰基团的周转速度都比母体分子快。它们还证明了一种新的再N-乙酰化反应，这预示着黑色素瘤细胞中存在脱N-乙酰神经节苷脂。
