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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571001/
• 关键词: HEMOLYTIC ANEMIA; MUTATION; PYRIMIDINE; RETICULOCYTES; ERYTHROCYTES; 赤血球酵素異常症; ピリミジン-5'-ヌクレオチダーゼ異常症; トランスジェニックマウス; インフルエンザウイルス; 免疫応答; 変異酵素; ミスセンス変異

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

本研究筛选了73例病因不明的非球型溶血性贫血患者，诊断为丙酮酸激酶缺陷5例、葡萄糖-6-磷酸脱氢酶缺陷10例、腺苷酸激酶缺陷1例、嘧啶5′-核苷酸酶缺陷1例。我们在9个P5N-I缺陷家族中发现了5个新的突变：2个错义突变（425C, 721C）， 1个剪接突变（339C）， 1个1 bp插入（251-insA-252）和1个9 bp缺失（del 192-200）。所有患者的每种突变都是纯合的。我们在Cos-7细胞中表达了带有425C和721C的突变体P5N-I，并检测了它们在细胞内的稳定性。L124P （425C）的剩余活性和免疫反应蛋白约为野生型对照的20%，表明L124P是一个不稳定的变体。G241R（721C）已被证明是一种对单磷酸胞苷亲和力较低的动力学变体。该突变体在Cos-7细胞中与对照一样稳定，与先前的结果一致。更多的结论表明Gly241对底物结合很重要。单倍型分析表明，在五个不相关的家庭中发现的721C是一个创始突变。由于蛋白酶体抑制剂恢复了L142P的稳定性，L142P增加了泛素-蛋白酶体途径降解的易感性。为了研究P5N-I在非红系细胞中的生理意义，我们建立了一个普遍过表达人P5N-I的转基因小鼠系。在肝脏和肾脏中均检测到转基因表达。然而，表达水平在正常对照的2倍以内。我们正在分析转基因P5N-I对肝脏和肾脏的影响。P5N-I基因可被α -干扰素诱导，在HIV感染或SLE等自身免疫性疾病患者的淋巴细胞中表达增强。从这些观察结果来看，p5n - 1被认为在免疫反应中有一定的作用。我们检测了P5N-I对流感感染急性免疫的影响；然而，P5N-I的过表达并没有改变感染细胞的免疫反应。进一步的研究将需要深入了解P5N-I的免疫学作用。我们感谢滨田孝子和冈田信一出色的技术支持。少

项目成果

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。

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

Murakami K, Kanno H, Tancabelic J, Fujii H: "Gene expression and biological significance of hexokinase in erythroid cells."Acta Haematol. 108. 204-209 (2002)

Murakami K、Kanno H、Tancabelic J、Fujii H：“红系细胞中己糖激酶的基因表达和生物学意义。”Acta Haematol。

Morimoto, A., Ueda, I., Kannno, H., Fujii, H., Imashuku S., et al.: "A novel missense mutation (1060G→C) in the phosphoglycerate kinase gene in a Japanese boy with chronic hemolytic anemia, developmental delay and rhabdomyolysis"Brit.J.Haematol. 122. 1009

Morimoto, A.、Ueda, I.、Kannno, H.、Fujii, H.、Imashuku S. 等人：“一名患有慢性溶血性贫血的日本男孩的磷酸甘油酸激酶基因中存在一种新的错义突变 (1060G→C)贫血、发育迟缓和横纹肌溶解症”Brit.J.Haematol. 122. 1009

Kanno, H., Murakami, K., Hariyama, Y., Ishikawa, K., Miwa, S., Fujii, H.: "Homozygous intragenic deletion of type-I hexokinase gene causes lethal hemolytic anemia of the affected fetus"Blood. 100(5). 1930 (2002)

Kanno, H.、Murakami, K.、Hariyama, Y.、Ishikawa, K.、Miwa, S.、Fujii, H.：“I 型己糖​​激酶基因的纯合基因内缺失导致受影响胎儿的致命性溶血性贫血”血液