Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells.

Using an expression cloning approach, we have isolated a cDNA encoding GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase, EC 2.4.99.8), which is a key regulatory enzyme determining the prominence of the ganglioside biosynthesis pathway. The cloned cDNA encodes a 341-amino acid protein containing a single transmembrane domain at its N-terminal region, suggesting that the protein has a type II transmembrane topology. The sequence of alpha 2,8-sialyltransferase showed a high level of similarity with other sialyltransferases at two conserved regions typical in the sialyltransferase family. Transfected cells containing the cloned cDNA expressed GD3 ganglioside on the cell surface, which was detectable with specific anti-GD3 antibody by immunofluorescence and immunostaining after separation of isolated glycolipids on thin-layer chromatography. The cDNA hybridized to a single mRNA species of 2.4 kb in melanoma cells. This sialyltransferase is distinctive in catalyzing the formation of the alpha 2-8 linkage of sialic acids. IMAGES:

本研究采用表达克隆法（expression cloning approach），分离得到了编码GD3合酶（GD3 synthase）的互补DNA（complementary DNA, cDNA）。该酶的系统命名为CMP-NeuAc:NeuAc α2-3Galβ1-4Glcβ1-1'Cer α2,8-唾液酸转移酶（α2,8-sialyltransferase, EC 2.4.99.8），是决定神经节苷脂生物合成通路活跃度的关键调控酶。所克隆的互补DNA编码一条含341个氨基酸残基的蛋白质，其N端区域包含单个跨膜结构域（transmembrane domain），提示该蛋白质具有II型跨膜拓扑结构（type II transmembrane topology）。该α2,8-唾液酸转移酶的序列，在唾液酸转移酶家族（sialyltransferase family）典型的两个保守区域中，与其他唾液酸转移酶呈现高度同源性。转染该克隆互补DNA的细胞可在细胞表面表达GD3神经节苷脂；通过薄层层析（thin-layer chromatography）分离纯化糖脂后，可利用特异性抗GD3抗体通过免疫荧光（immunofluorescence）及免疫染色（immunostaining）检测到该产物。该互补DNA可与黑色素瘤细胞（melanoma cells）中一条2.4 kb的单一信使RNA（messenger RNA, mRNA）转录本发生杂交。该唾液酸转移酶的独特之处在于其催化唾液酸α2,8-糖苷键的形成。图像：