STRUCTURE AND FUNCTION OF NOVEL AMINOPEPTIDASES BELONGING TO OXYTOCINASE SUB-FAMILY

属于催产酶亚家族的新型氨肽酶的结构和功能

• 批准号: 11470492
• 负责人: TSUJIMOTO Masafumi
• 金额: $ 4.16万
• 依托单位: Riken
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470492/
• 关键词: AMINOPEPTIDASE; OXYTOCINASE; PLACENTAL LEUCINE AMINOPEPTIDASE; ADIPOCYTE-DERIVED LEUCINE AMINBOPEPTIDASE; BIOACTIVE PEPTIDE; VASOPRESSIN; BLOOD PRESSURE; cDNA CLONING; 尿細管; プロモーター領域; 神経細胞; ペプチドホルモン; CHO細胞; タンパク質結晶化; 金属酵素; 胎盤性ロイシンアミノペプチターゼ

It is well known that placental leucine aminopeptidase (P-LAP)/oxytocinase increases in matemal serum during pregnancy and maintaihs normal pregnancy by preventing the action of oxytocin. We have cloned the cDNA encoding P-LAP for the first time and shown that the enzyme is a type II membrane- spanning protein. In this study we have further analysed pathological/physiological properties of the eazyme in detail.We have constructed a large-scale production system of recombinant P-LAP employing CHO cells and analysed the enzymatic properties of the enzyme. It was found that in addition to oxytocin and vasopressin, the enayme degraded some neuronal peptides such as Met-enkephalin and dynorphin A. Since the eazyme was expressed in neuronal cells and increased in its expression during neuronal differentiation, it is considered that P-LAP plays an important role in the regulation of neuronal cells in the brain. Since antidiuretic hormone vasopressin, a good substrate for P-LAP, regulates wate … More r reabsorption in renal collecting duct principal cells and expression of P-LAP in the kidne was detected, we have analysed the effect of the hormone on the translocation of the eazyme rom intracellular vesicle to plasma membrane. It was found that after treatment of NRK52E cells with vasopressin, rapid translocation was clearly detected. These results suggested the existence of the P-LAP mediated negative feedback mechabism of the vasopressin action in the kidney. Moreover we have analysed the promoter function of P-LAP gene and found that twotranscription factors, AP-2 and ikaros, were important for the regulation of the gene.By searching a database, we have cloned a novel enzyme tenned adipocyte-derived leucine aminopeptidase (A-LAP). Detailed structural analysis indicated that P-LAP and A-LAP should be classified into oxyiocinase sub-fatnily. Enzymatic analysis after construcdon of a large-scale production system of recombinant A-LAP suggested that the enzyme played a role in the regulation of blood pressure through the inactivation of angiotensin 11 and/or the generation of bradyki' nin in the kidney., Less

妊娠期母体血清中胎盘亮氨酸氨基肽酶(P-LAP)/催产素酶升高，通过阻止催产素的作用维持正常妊娠。我们首次克隆了编码P-LAP的cDNA，并证实该酶是II型跨膜蛋白。在这项研究中，我们进一步详细分析了酶的病理/生理特性。我们利用CHO细胞构建了重组P-LAP的大规模生产体系，并分析了该酶的酶学性质。研究发现，除了催产素和抗利尿激素外，酶还能降解一些神经肽，如Met-enkephalin和dynorphin a。由于酶在神经元细胞中表达，并在神经元分化过程中表达增加，因此认为P-LAP在脑内对神经元细胞的调节中起着重要作用。由于抗利尿激素加压素是P-LAP的良好底物，可以调节肾集管主细胞的水分重吸收和P-LAP在肾脏中的表达，我们分析了该激素对酶从胞内囊泡向质膜转运的影响。我们发现，加压素处理NRK52E细胞后，可以明显检测到快速易位。这些结果提示肾内抗利尿激素作用存在P-LAP介导的负反馈机制。此外，我们还分析了P-LAP基因的启动子功能，发现两个转录因子AP-2和ikaros在该基因的调控中起重要作用。通过检索数据库，我们克隆了一种名为脂肪细胞衍生亮氨酸氨基肽酶（a - lap）的新酶。详细的结构分析表明，P-LAP和A-LAP应归为氧辛酸酶亚系。重组a - lap大规模生产系统构建后的酶学分析表明，该酶通过使血管紧张素11失活和/或在肾脏中产生bradyki' nin来调节血压。,更少

项目成果

Yamahara N.et al.: "Placental leucine aminopeptidase/Oxytocinase in maternal serum and placenta during normal pregnancy"Life Science. 66. 1401-1410 (2000)

Yamahara N.等人：“正常妊娠期间母体血清和胎盘中的胎盘亮氨酸氨肽酶/催产素酶”生命科学。

Hattori A., et al.: "Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase oxytocinase"J. Biochem.. 125. 931-938 (1999)

Hattori A.等人：“与胎盘亮氨酸氨肽酶催产素酶高度相关的脂肪细胞来源的亮氨酸氨肽酶的分子克隆”J.

Matsumoto H., et al.: "Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells"Eur. J. Biochem.. 267. 46-52 (2000)

Matsumoto H.等人：“在中国仓鼠卵巢细胞中表达的人胎盘亮氨酸氨基肽酶/催产素酶的重组可溶形式的表征”Eur.

Hattori A, et al.: "Genomic organization of the human adipocyte-derived leucine aminopeptidase gene and its relationship to the placental leucine aminopeptidase/oxytocinase gene"J. Biochem.. 130. 235-241 (2001)

Hattori A 等人：“人脂肪细胞来源的亮氨酸氨肽酶基因的基因组结构及其与胎盘亮氨酸氨肽酶/催产素酶基因的关系”J.

Katsumata Y., et al.: "Progesterone stimulates the expression of aminopeptidase A/angiotensinase in human choriocarcinoma cells"Placenta. 22. 831-836 (2001)

Katsumata Y. 等人：“黄体酮刺激人绒毛膜癌细胞中氨肽酶 A/血管紧张素酶的表达”胎盘。