MOLECULAR BASIS OF PYRIMIDINE 5'-NUCLEOTIDASE (P5N) DEFICIENCY AND FUNCTIONAL ANALYSIS OF P5N

嘧啶 5-核苷酸酶 (P5N) 缺陷的分子基础及 P5N 的功能分析

基本信息

批准号：
14571001
负责人：
FUJII Hisaichi
金额：
$ 2.18万
依托单位：
TOKYO WOMEN'S MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

In this study, we screened 73 subjects with non-spherocytic hemolytic anemia of unknown pathogenesis, and diagnosed 5 of pyruvate kinase, 10 of glucose-6-phosphate dehydrogenase, 1 of adenylate kinase and 1 of pyrimidine 5'-nucleotidase (P5N-I) deficient cases.We identified five novel mutations in nine families with P5N-I deficiency : two missense mutations (425C, 721C), one splicing mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients are homozygous for each mutation.We expressed mutant P5N-I with 425C and 721C in Cos-7 cells, and examined their intracellular stability. The L124P (425C) showed approximately 20% residual activity and immunoreactive protein of a wild-type control, suggesting that the L124P was an unstable variant. The G241R(721C) had been demonstrated as a kinetic variant with lower affinity for cytidine monophosphate. This mutant was as stable as a control in Cos-7 cells, being compatible with previous results. We could c … More onclude that Gly241 is important for the substrate binding. Haplotype analysis showed that the 721C, which had been identified in five unrelated families, was a founder mutation. Since proteasome inhibitors restored the stability of the L142P, the L142P increases the susceptibility to the degradation by ubiquitin-proteasome pathway.To examine physiological significance of P5N-I in non-erythroid cells, we established a line of transgenic mouse, which ubiquitously overexpressed human P5N-I. Transgene expression was detected in both liver and kidney. However, expression levels are within 2 times of normal controls. Effects of transgene-derived P5N-I on liver and kidney are being analyzed.The P5N-I gene can be induced by alpha-interferon, and the expression is augmented in lymphocytes of patients with HIV infection or autoimmune diseases such as SLE. From these observations, the P5N-I has been suggested to have any roles on immune response. We examined effects of the P5N-I on acute immunity of influenza infection ; however, overexpression of the P5N-I did not modify immune responses of infected cells. Further studies will require gaining insights on immunological roles of P5N-I. We thank Takako Hamada and Shin-ichi Okada for their excellent technical supports. Less

In this study, we screened 73 subjects with non-sphere-hemolytic anemia of unknown pathogenesis, and diagnosed 5 of pyruvate kinase, 10 of glucose-6-phosphate dehydrogenase, 1 of adenylate kinase and 1 of pyrimidine 5'-nucleotidase (P5N-I) deficient cases.We identified five novel mutations in nine families with P5N-I deficiency : two错义突变（425c，721c），一个剪接突变（339c），一个1 bp插入（251-INSA-252）和一个9 bp缺失（DEL 192-200）。所有患者在每个突变中都是纯合的。我们在COS-7细胞中用425C和721C表达突变体P5N-I，并检查了其细胞内稳定性。 L124p（425C）的残留活性约为20％，并具有野生型对照的免疫反应性蛋白质，这表明L124p是不稳定的变体。 G241R（721C）已被证明是一种对胞苷单磷酸的亲和力较低的动力学变体。该突变体与COS-7细胞中的对照一样稳定，与先前的结果兼容。我们可以……更多地说，Gly241对于底物结合很重要。单倍型分析表明，在五个无关家族中已鉴定出的721C是一个创始人突变。由于蛋白酶体抑制剂恢复了L142P的稳定性，因此L142P增加了泛素蛋白蛋白蛋白酶体途径降解的敏感性。要检查p5n-i在非雌激素细胞中的物理意义，我们在非毛细血管中的物理意义，我们建立了一种转基因小鼠的线，这些小鼠是ubiquicalliquicallicalliquicalliquicalliquicalliquicalliquicallicalliquicalliquicalliquicallicalliperapialed expercaced holduts p5n-i。在肝脏和肾脏中都检测到转基因表达。但是，表达水平在正常对照的2倍之内。转基因衍生的P5N-I对肝脏和肾脏的影响正在分析中。p5N-I基因可以由α-I-I-I-I-I-I基因诱导，并且在HIV感染或自身免疫性疾病的患者的淋巴细胞中增强了表达。从这些观察结果中，已经提出P5N-I在免疫响应中起任何作用。我们检查了P5N-I对影响力感染的急性免疫学的影响；但是，P5N-I的过表达不会改变感染细胞的免疫血液。进一步的研究将需要对P5N-I的免疫学作用有所了解。我们感谢Takako Hamada和Shin-ichi Okada的出色技术支持。较少的

项目成果

期刊论文数量（34）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Kanno, H., Fujii, H., Miwa, S.: "Physiological significance and molecular genetics of red cell enzymes involved in the ribonucleotide metabolism"Proc. Japan Acad. 78(10). 287-292 (2002)

Kanno，H.，Fujii，H.，Miwa，S.：“参与核糖核苷酸代谢的红细胞酶的生理意义和分子遗传学”Proc。

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Morimoto A, Ueda I, Hirashima Y, Sawai Y, Usuku T, Kano G, Kuriyama K, Todo S, Sugimoto T, Kanno H, Fujii H, Imashuku S: "A novel missense mutation (1060G→C) in the phosphoglycerate kinase gene in a Japanese boy with chronic hemolytic anemia, developmenta

Morimoto A、Ueda I、Hirashima Y、Sawai Y、Usuku T、Kano G、Kuriyama K、Todo S、Sugimoto T、Kanno H、Fujii H、Imashuku S：“磷酸甘油酸激酶中的一种新型错义突变 (1060G→C)患有慢性溶血性贫血的日本男孩的基因，正在发育