Study on structures and functions of CMP-sialic acid modifying enzymes

本研究的目的是分离与糖脂合成有关的哺乳动物O-乙酰转移酶编码基因。有两种分离cDNA的方法：(I)基于从纯化的蛋白质或酶抗体获得的序列信息的方法；(Ii)不需要肽序列的方法。在第一年，我们检查了哪种程序更适合分离cDNA。我们发现：(1)转移酶在膜组分中丰富；(2)用纯化的神经节苷脂免疫小鼠所建立的小鼠特异性单抗(MAb)可使几种人黑色素瘤细胞系大量地表达O-Ac-GD3。基于这些结果，我们决定采用一种哺乳动物的cDNA表达克隆方案。作为下一步，我们测定了一些宿主细胞的糖脂结构和糖基转移酶的活性。两个细胞系(COS-1和COP5)是候选宿主细胞。这些细胞均不表达O-Ac-GD3。此外，COS-1细胞表达多种神经节苷脂，包括GM3和GD3。因此，选择COS-1细胞作为宿主细胞。利用B.Seed方法和哺乳动物表达载体pCDM8，从表达大量相关神经节苷脂的人肿瘤细胞中构建了CDNA文库。用该质粒文库转移细胞。采用DEAE-葡聚糖转染法。将转基因细胞加入含有吸附抗体的培养皿中，采用平皿技术。我们一直在尝试用单抗分离阳性细胞。

The aim of this research was to isolate cDNAs encoding mammalian O-Acetyltransferases involved in the synthsis of glycolipids. There are two methods for the isolation of the cDNAs ; (i) an approach that based on sequence information derived from the purified protein or antibodies to the enzymes ; (ii) an approach that does not require sequence of the peptides. In the first year, we checked which procedure is better for the isolation of the cDNAs. We found that (i) the transferase was enriched in the membrane fraction, (ii) several human melanoma cell lines empressed a large amount of O-Ac-GD3 with a specific mouse monoclonal antibody (MAb), which was established by immunizing mice with the purified ganglioside. Based on these results, we have decided to adapt an mammalian cDNA expression cloning scheme. As a next step, we determined the structure of glycolipids and the activities of the glycosyltransferase from a number of host cells. Two cell lines (COS-1 and COP5) were candidates for the host cells. None of these cells expressed O-Ac-GD3. w Moreover, COS-1 cells expressed a number of gangliosides, including GM3 and GD3. Thus, COS-1 cells were selected as a host cells. A CDNA library was prepared from human tumor cells that express a large amount of relevant gangliosides on cell surfaces, using the procedure of B. Seed and the mammalian expression vector pCDM8. Cells were transfered with the plasmid library. The DEAE-dextran method of transfection was used. The transfected cells were added to dishes containing absorbed antibody following the panning technique. We have been trying to isolate positive-cells with the MAb.