Analysis of two types of sialytransferases involved in polysialic acid synthesis

在N-CAM中发现了聚ailic酸，其表达具有器官特异性，并受到严格控制。我们克隆了两个不同的a2,8-唾液基转移酶（STX和PST），它们直接参与了聚唾液酸的生物合成。然而，目前尚不清楚这些酶是如何参与控制聚唾液酸的表达的。为了了解聚唾液酸在体内的控制，本研究澄清了以下问题。1)鉴定小鼠PST基因的基因组结构，分析其启动子区域。结果表明，小鼠PST基因的基因组结构与小鼠STX相似，但小鼠PST基因启动子区域的转录基序与小鼠STX基因完全不同。因此，两种聚唾液酸合酶的表达似乎是独立控制的。2)将小鼠STX基因转染到不表达聚唾液酸但表达N-CAM的Neuro2a细胞中，N-CAM 140和N-CAM 180被选择性地多唾液酸化。另一方面，在PST基因转染的小鼠神经2a细胞中，其他糖蛋白也被多唾液化。因此，体内STX的底物特异性与PST不同。3)将小鼠STX或PST基因转染到不能合成含焦寡糖的CHO突变细胞系Lec13中，聚唾液酸在Lec13中的表达量大大低于亲本CHO。通过对Lec13细胞中低聚糖的结构分析和脉冲追踪实验表明，聚唾液酸在Lec13细胞中的表达降低是由于Lec13细胞中聚唾液化、非聚焦化的糖蛋白降解所致。

Polysailic acid has been found in N-CAM, and its expression has been known to be organ-specific and to be critically controlled. We cloned two different a2,8-sialyltransferases (STX and PST), which were directly involved in the biosynthesis of polysialic acid. However, it is still unclear how these enzymes are involved in controlling of the expression of polysialic acid. In this study, following pits were clarified in order to understand the control of polysialic acid in vivo.1) The genomic structure of mouse PST gene was identified and its promoter region was analyzed. The results indicated that the genomic structure of mouse PST gene was similar to that of mouse STX, but the transcriptional motifs in the promoter region of mouse PST gene was completely different from those of mouse STX gene. Therefore, the expression of two polysialic acid synthases was seemed to be independently controlled.2) When mouse STX gene was transfected into Neuro2a cells, which did not expressed polysialic acid but did N-CAM, N-CAM 140 and N-CAM 180 were selectively polysialylated. On the other hand, other glycoproteins were also polysialylated in mouse PST gene-transfected Neuro2a cells. Therefore, the substrate specificity of STX was different from that of PST in vivo.3) When mouse STX or PST gene was transfected into CHO mutant cell line, Lec13, which could not synthesize fucose-containing oligosaccharides, the expression of polysialic acid in Lec13 was greatly lower than that in the parent CHO. The structural analysis of oligosaccharides in Lec13 cells and pulse-chase experiments indicated that decreasing of the expression of polysialic acid in Lec13 cells was due to the degradation of polysialylated, non-fucosylated glycorteins in Lec13 cells.