Structure and Function of the Phospholipase AィイD22ィエD2 Inhibitor Having Leucine-rich Repeats

富含亮氨酸重复序列的磷脂酶 AD22 抑制剂的结构和功能

• 批准号: 10672074
• 负责人: INOUE Seiji
• 金额: $ 2.37万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672074/
• 关键词: Phospholipase AィイD22ィエD2; Inhibitor; Venomous snake; Leucine-rich repeats; Tertiary structure; Serum; cDNA; Inhibition mechanism; 毒ヘビ

1. A large-scale purification of phospholipase AィイD22ィエD2 inhibitors from the serum of Chinese mamushi and the crystallization of the inhibitorsWe have purified three types of phospholipase AィイD22ィエD2 inhibitors (PLIα, PLIβ, and PLIγ) by the sequential chromatography on Blue-Sepharose FF, Q-Sepharose FF, Phenyl-Sepharose HP, and Superdex 200pg columns. Finally, 10 mg of the purified PLIα, 5 mg of the PLIβ, and 20 mg of the PLIγ could be obtained from 10 ml of the Chinese mamushi serum. We are determining the best crystallization conditions of these inhibitors by the standard technique of vapor diffusion with the hanging drop methods. By this time, we could not obtain the crystals suitable for X-ray crystallography.2. Establishment of the bacterial expression system for PLAィイD22ィエD2 inhibitors and elucidation of their structure function relationships by means of the site-directed mutagenesis analysisThe PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Esc … More herichia coli. We could obtain the recombinant protein having the inhibition activity as strong as that of the natural PLIα. Alternatively, we have identified a PLIα-like protein (PLIα-LP) from the serum of the non-venomous striated snake and cloned its cDNA. The purified PLIα-LP was found to have no inhibitory activity toward any PLAィイD22sィエD2, although its amino acid sequence showed about 70% homology to that of Chinese mamushi PLIα. We have constructed the recombinant chimeric proteins between the PLIα-LP and Chinese mamushi PLIα and examined their inhibitory activity in order to specify the region responsible for the PLAィイD22ィエD2 inhibition. It was suggested that the CRD structure in the PLIα molecule played the basic structural roles such as the formation of trimeric structure and that the N-terminal and C-terminal regions of PLIα other than CRD were required for the interaction of PLIα to a PLAィイD22ィエD2 molecule.3. Purification of leucine-rich αィイD22ィエD2 glycoprotein (LRG) from the human serum and the examination of its PLAィイD22ィエD2 inhibitory activity.Using the synthesized peptide (LRG1-20) corresponding to the reported N-terminal 20 amino acid sequence of LRG as an antigen, we immunized a rabbit and obtained the anti-LRG1-20 antibody. The ELISA using this antibody was found to be a good tool for detecting LRG during the purification of LRG from human serum. Less

1. 采用Blue-Sepharose FF、Q-Sepharose FF、Phenyl-Sepharose HP和Superdex 200pg色谱柱，对3种磷脂酶（PLIα、PLIβ和PLIγ）抑制剂进行了纯化。最后，从10 ml中国马木鱼血清中可得到纯化的PLIα 10 mg、PLIβ 5 mg和PLIγ 20 mg。我们用悬挂滴法的标准蒸汽扩散技术确定了这些抑制剂的最佳结晶条件。此时，我们还不能得到适合x射线晶体学的晶体。建立细菌表达系统的解放军ィイD22摊位ィエD2抑制剂和说明其结构功能的关系通过定点诱变analysisThe PLIαcDNA subcloned进入表达载体pET-16b和用于转换Esc ... 更多的herichia杆菌。我们可以获得与天然PLIα具有相同抑制活性的重组蛋白。另外，我们从无毒条纹蛇的血清中鉴定出一种pli α-样蛋白（PLIα-LP），并克隆了其cDNA。纯化后的PLIα- lp对PLA γ γ γ γ - 22和γ γ - D2均无抑制活性，但其氨基酸序列与中国马木鱼PLIα同源性约为70%。我们构建了PLIα- lp与中国马马鱼PLIα之间的重组嵌合蛋白，并检测了它们的抑制活性，以确定抑制PLA γ γ γ γ - D22 γ γ - D2的区域。建议PLIαCRD结构分子的基本结构等角色的形成三聚物的结构和PLIα的氨基端和c端区域交互所需的其他比CRD PLIα的解放军ィイD22摊位ィエD2 molecule.3。人血清中富含亮氨酸的α γ γ γ - D22 γ - D2糖蛋白（LRG）的纯化及其对PLA γ - D22 γ - D2抑制活性的检测。利用合成的LRG n端20氨基酸序列对应的肽段（LRG1-20）作为抗原免疫家兔，获得抗LRG1-20抗体。在人血清中LRG的纯化过程中，使用该抗体的ELISA检测是一种较好的工具。少

项目成果

Okumura, K., Inoue, S., Ohkura, N., Ikeda, K., and Hayashi, K.: "cDNA Cloning of the Two Subunits of Phospholipase AィイD22ィエD2 Inhibitor PLIγ from Blood Plasma of the Chinese Mamushi, Agkistrodon blomhoffii siniticus"IUBMB Life. 48 (1). 99-104 (1999)

Okumura, K.、Inoue, S.、Ohkura, N.、Ikeda, K. 和 Hayashi, K.：“从中国 Mamushi、Agkistrodon blomhoffii siniticus 血浆中克隆磷脂酶 AiiD22IeD2 抑制剂 PLIγ 的两个亚基的 cDNA” IUBMB 生活。48 (1)。

Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K.: "cDNA cloning and Bacterial Expression of Phospholipase AィイD22ィエD2 Inhibitor PLIα from the Serum of the Chinese Mamushi, Agkistrodon blomhoffii siniticus"Biochim. Biophys. Acta.. 1441 (1). 51-60 (1999)

Okumura, K.、Inoue, S.、Ikeda, K. 和 Hayashi, K.：“来自中国 Mamushi、Agkistrodon blomhoffii siniticus 血清的磷脂酶 A-D22-D2 抑制剂 PLIα 的 cDNA 克隆和细菌表达”Biochim。生物物理学报。1441（1）。

Ohkura, N., Kitahara, Y., Inoue, S., Ikeda, K., and Hayahsi, K.: "Isolation and Amino Acid Sequence of a Phospholipase AィイD22ィエD2 inhibitor from the Blood Plasma of the Sea Krait, Laticauda semifasciata"J. Biochem.. 125 (2). 375-382 (1999)

Ohkura, N.、Kitahara, Y.、Inoue, S.、Ikeda, K. 和 Hayahsi, K.：“从海蛇血浆中分离出磷脂酶 A-D22-D2 抑制剂和氨基酸序列， Laticauda semifasciata”J.Biochem.125(2).375-382(1999)

Okumura,K.: "cDNA Cloning of the Two Subunits of Phospholipase A_2 Inhibitor PLIγ form Blood Plasma of the Chinese Mamushi, Agkistrodon blomhoffii siniticus"IUBMB Life. 48(1). 99-104 (1999)

Okumura, K.：“中国 Mamushi、Agkistrodon blomhoffii siniticus 血浆中磷脂酶 A_2 抑制剂 PLIγ 的两个亚基的 cDNA 克隆”IUBMB Life 48(1)。

Okumura, K.: "Purification, Characterization and cDNA Cloning of a Phospholipase A_2 Inhibitor from the Serum of the Non-venomous snake Elaphe quadrivirgata"Biochem. J.. 341(1). 165-171 (1999)

Okumura, K.：“无毒蛇 Elaphequadrivirgata 血清中磷脂酶 A_2 抑制剂的纯化、表征和 cDNA 克隆”Biochem。