Analysis of multi-function of renin binding protein

肾素结合蛋白的多功能分析

• 批准号: 16580082
• 负责人: TAKAHASHI Saori
• 金额: $ 2.37万
• 依托单位: Akita Research Institute of Food and Brewing
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580082/
• 关键词: renin; binding protein; nucleotides; ATP; N-acetyl-D-mannosamine; epimerase; multi-functional protein; 多機能タンパク質; 部位特異的変異体; 高血圧

Renin binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. Recently the protein was identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The enzyme catalyzes the interconversion between GlcNAc and N-acetyl-D-mannosamine (ManNAc) and nucleotide are essential as a cofactor. Our recent studies demonstrated that the middle domain of GlcNAc 2-epimerase participates in the specificity for and binding of nucleotide. To identify the residue conferring nucleotide binding, amino acid substitutions were introduced in the human and rat GlcNAc 2-epimerases. The expression and characterization of the mutants indicate that reside 171 of GlcNAc 2-epimerase is critical for the nucleotide binding of the enzyme. The recombinant porcine RnBP inhibited porcine renin activity in a dose dependent manner in the absence of nucleotide. But the inhibition was neutralized by nucleotides such as ATP,dATP,dGTP,dCTP, or dTTP. ATP inhibited the formation of hetero-complex of renin with RnBP. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited GlcNAc 2-epimerase activity concomitant decaying dimer to monomer of the enzyme. NEM did not inhibit GlcNAc 2-epimerase activity in the presence of ATP. These results clearly indicate that nucleotides stabilize dimeric from of RnBP (GlcNAc 2-epimerase) and inhibited the formation of renin-RnBP hetero-complex, HMW renin. Moreover, the human renin was expressed in E. coli cells as a fusion protein of thioredoxin. The chimeric protein, accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by L-arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the renin activity. The active recombinant human renin will be used for the physiological studies of human RnBP in near future.

肾素结合蛋白（Renin binding protein，RnBP）是一种来源于猪肾的内源性肾素抑制剂。最近，该蛋白被鉴定为酶N-乙酰基-D-葡糖胺（GlcNAc）2-差向异构酶。该酶催化GlcNAc和N-乙酰基-D-甘露糖胺（ManNAc）之间的相互转化，并且核苷酸作为辅因子是必需的。我们最近的研究表明，GlcNAc 2-差向异构酶的中间结构域参与了核苷酸的特异性结合。为了鉴定赋予核苷酸结合的残基，在人和大鼠GlcNAc 2-差向异构酶中引入氨基酸取代。突变体的表达和表征表明GlcNAc 2-差向异构酶的171位残基对于酶的核苷酸结合是关键的。重组猪RnBP在核苷酸不存在的情况下以剂量依赖的方式抑制猪肾素活性。而ATP、dATP、dGTP、dCTP、dTTP等核苷酸能中和这种抑制作用。ATP抑制RnBP与肾素形成异源复合物。另一方面，N-乙基马来酰亚胺（NEM），SH-烷基化试剂抑制GlcNAc 2-差向异构酶活性，伴随着酶的二聚体衰变为单体。NEM在ATP存在下不抑制GlcNAc 2-差向异构酶活性。这些结果清楚地表明，核苷酸稳定RnBP（GlcNAc 2-差向异构酶）的二聚体形式，并抑制肾素-RnBP异源复合物HMW肾素的形成。此外，在E.大肠杆菌细胞作为硫氧还蛋白的融合蛋白。嵌合蛋白在4 M盐酸胍中溶解后，经L-精氨酸-去污剂缓冲液系统和系统透析复性。复性后的融合原肾素被胰蛋白酶激活。抗人肾肾素抗血清能特异性地抑制肾素活性。具有活性的重组人肾素将在不久的将来用于人RnBP的生理研究。

项目成果

Amino acid residues conferring nucleotide binding properties of N-acetyl-D-glucosamine 2-epimerase (renin binding protein).

氨基酸残基赋予 N-乙酰基-D-葡萄糖胺 2-差向异构酶（肾素结合蛋白）的核苷酸结合特性。

• 发表时间: 2005
• 期刊: Biomedical Research 26(3)
• 作者: Saori Takahashi;Hironobu Ogasawara;Keishi Hata;Kazuyuki Hiwatashi;Kazuyuki Hori;Saori Takahashi et al.
• 通讯作者: Saori Takahashi et al.

「研究成果報告書概要(欧文)」より

摘自《研究结果报告摘要（欧洲）》

• 发表时间: 2006
• 期刊: Seibutsu Butsuri 46(1)
• 作者: Yasushi Shigeri;Keiko Shimamoto
• 通讯作者: Keiko Shimamoto

Advances in Chitin Science Vol.VIII

甲壳素科学进展第八卷

• 发表时间: 2005
• 作者: Saori Takahashi;Hironobu Ogasawara;Keishi Hata;Kazuyuki Hiwatashi;Kazuyuki Hori;Saori Takahashi et al.;Saori Takahashi et al.;Saori Takahashi et al.;Saori Takahashi et al.
• 通讯作者: Saori Takahashi et al.

Advances in Chitin Science Vol.8

甲壳素科学进展第 8 卷

• 发表时间: 2005
• 作者: Saori Takahashi;Hironobu Ogasawara;Keishi Hata;Kazuyuki Hiwatashi;Kazuyuki Hori;Saori Takahashi et al.;Saori Takahashi et al.;Saori Takahashi et al.
• 通讯作者: Saori Takahashi et al.

Amino acid residues conferring nucleotide binding properties of N-acetyl-D-mannosamine 2-epimerase (renin binding protein)

赋予 N-乙酰基-D-甘露糖胺 2-差向异构酶（肾素结合蛋白）核苷酸结合特性的氨基酸残基

• 发表时间: 2005
• 期刊: Biomedical Research 26(3)
• 作者: Nozaki;K.;et al.;H.Ito et al.;N.Isono et al.;K.Nozaki et al.;T.Senoura et al.;Saori Takahashi et al.
• 通讯作者: Saori Takahashi et al.