|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥1,600,000 (Direct Cost : ¥1,600,000)
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.