Molecular and Functional Analysis of Heparan sulfate Proteoglycan-Ryudocan.

Ryudocan是一种普遍存在的硫酸肝素蛋白聚糖，是syndecan细胞表面蛋白聚糖家族的成员。目前已克隆并测序了编码鼠核蛋白的全长cDNA。推导出的小鼠ryudocan的初级结构，包括胞外区域以及跨膜和细胞质区域的三个糖胺聚糖附着位点，与大鼠、人类和鸡的蛋白质高度相似。Northern分析在所有小鼠组织中检测到一个2.7 kb的转录本，在肝脏、肾脏和肺中浓度最高。小鼠的ryudocan基因跨越大约19.7 kb的基因组DNA，包含5个外显子，内含子-外显子的结构与人类基因相同。小鼠基因的启动子区域包含多种顺式作用元件，包括tata样盒和GC盒，以及转录因子NF-IL6、MyoD、GATA、C/EBP、AP-2、NF-kB、AP-1和Sp1的潜在结合位点。瞬时转染实验显示，转录起始位点上游690bp的构建体与荧光素酶报告基因融合，显示出功能性启动子活性。缺失分析表明，近端启动子区域包括TATA-like box、GC box和其他Sp1结合位点是完全转录活性所必需的。这些发现将有助于研究琉球can基因调控和产生靶向破坏该基因的小鼠。

Ryudocan, a ubiquitous heparan sulfate proteoglycan, is a member of the syndecan family of cell surface proteoglycans. The full-length cDNA encoding the murine ryudocan core protein has now been cloned and sequenced. The deduced primary structure of mouse ryudocan, including the three glycosaminoglycan attachment sites in the extracellular domain as well as the transmembrane and cytoplasmic regions, is highly similar to those of the rat, human, and chicken proteins. Northern analysis detected a 2.7-kb transcript in all mouse tissues examined, with the highest concentrations apparent in liver, kidney, and lung. The mouse ryudocan gene was shown to span approximately 19.7 kb of genomic DNA and to contain five exons, with an intron-exon organization identical to that of the human gene. The promoter region of the mouse gene contains various cis-acting elements, including a TATA-like box and GC box as well as potential binding sites for the transcription factors NF-IL6, MyoD,GATA,C/EBP,AP-2, NF-kB,AP-1, and Sp1. Transient transfection experiments with a construct containing the 690 bp upstream of the transcription start site fused to a luciferase reporter gene showed functional promoter activity. Deletion analysis suggested that the proximal promoter region including the TATA-like box, GC box, and other Sp1 binding sites was required for full transcriptional activity. These findings will be useful for the study of ryudocan gene regulation and the generation of mice with targeted disruption of the gene.