Kopitz J, von Reitzenstein C, Sinz K, Cantz M
Institute of Pathochemistry and General Neurochemistry, University of Heidelberg, Germany.
Glycobiology. 1996 Apr;6(3):367-76. doi: 10.1093/glycob/6.3.367.
Gangliosides of the plasma membrane are important modulators of cellular functions. Previous work from our laboratory had suggested that a plasma membrane sialidase was involved in growth control and differentiation in cultured human neuroblastoma cells (SK-N-MC), but its substrates had remained obscure. We now performed sialidase specificity studies in subcellular fractions and found ganglioside GM3 desialylating activity in presence of Triton X-100 to be associated with the plasma membrane, but absent in lysosomes. This Triton-activated plasma membrane enzyme desialylated also gangliosides GD1a, GD1b, and GT1b, thereby forming GM1; cleavage of GM1 and GM2, however, was not observed. Sialidase activity towards the glycoprotein fetuin with modified C-7 sialic acids and towards 4-methylumbelliferyl neuraminate was solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in gangliosides desialylation of living cells was examined by following the fate of [3H]galactose-labelled individual gangliosides in pulse-chase experiments in absence and presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. When the plasma membrane sialidase was inhibited, radioactivity of all gangliosides chased at the same rate. In the absence of inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degraded at a considerably faster rate in confluent cultures, whereas the GM1-pool seemed to be filled by the desialylation of higher gangliosides. The results thus suggest that the plasma membrane sialidase causes selective ganglioside desialylation, and that such surface glycolipid modification triggers growth control and differentiation in human neuroblastoma cells.
质膜神经节苷脂是细胞功能的重要调节因子。我们实验室之前的研究表明，一种质膜唾液酸酶参与了培养的人神经母细胞瘤细胞（SK-N-MC）的生长控制和分化，但其底物仍不清楚。我们现在在亚细胞组分中进行了唾液酸酶特异性研究，发现在存在Triton X-100的情况下，神经节苷脂GM3的去唾液酸化活性与质膜相关，但在溶酶体中不存在。这种Triton激活的质膜酶也能使神经节苷脂GD1a、GD1b和GT1b去唾液酸化，从而形成GM1；然而，未观察到GM1和GM2的裂解。对具有修饰的C-7唾液酸的糖蛋白胎球蛋白和对4-甲基伞形酮基神经氨酸的唾液酸酶活性仅在溶酶体中发现，而在质膜组分中未发现。通过在有无细胞外唾液酸酶抑制剂2-脱氧-2,3-脱氢-N-乙酰神经氨酸的脉冲追踪实验中追踪[3H]半乳糖标记的单个神经节苷脂的命运，研究了质膜唾液酸酶在活细胞神经节苷脂去唾液酸化中的作用。当质膜唾液酸酶被抑制时，所有神经节苷脂的放射性以相同的速率追踪。在没有抑制剂的情况下，GM3、GD1a、GD1b、GD2、GD3和GT1b在汇合培养物中以相当快的速度降解，而GM1池似乎由较高神经节苷脂的去唾液酸化填充。因此，结果表明质膜唾液酸酶导致选择性神经节苷脂去唾液酸化，并且这种表面糖脂修饰触发人神经母细胞瘤细胞的生长控制和分化。
