Haraguchi M, Yamashiro S, Yamamoto A, Furukawa K, Takamiya K, Lloyd K O, Shiku H, Furukawa K
Department of Oncology, Nagasaki University School of Medicine, Japan.
Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10455-9. doi: 10.1073/pnas.91.22.10455.
For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.
为了分离神经节苷脂GD3合酶（EC 2.4.99.8）的cDNA，我们开发了一种表达克隆方法，该方法使用抗GD2单克隆抗体进行筛选。我们还利用了一种命名为KF3027-Hyg5的宿主受体细胞系。该细胞系表达高水平的GM2以及GM3，但不表达GD3或GD2，它是由转染了多瘤大肿瘤抗原基因（KF3027）和先前克隆的β-1,4-N-乙酰半乳糖胺基转移酶（EC 2.4.1.92）cDNA的小鼠B16黑色素瘤细胞构建而成。经过四轮转染、单克隆抗体3F8淘选和Hirt提取，分离出了两个cDNA克隆，将其转染可使KF3027和B16黑色素瘤细胞中表达GD3，在KF3027-Hyg5细胞中表达GD3和GD2。该cDNA包含一个1650 bp的插入片段和一个单一的开放阅读框。推导的氨基酸序列预测其为II型膜拓扑结构，由胞质结构域（14个氨基酸）、跨膜结构域（18个氨基酸）和催化结构域（309个氨基酸）组成。该序列还预测存在一个与迄今克隆的其他唾液酸转移酶中发现的唾液酸基序相似的基序。正如预期的那样，该基因的mRNA（2.6 kb）在人黑色素瘤细胞系中强烈表达。
