Biochemical characterization of human CLN2, related to a fatal neurodegenerative disease : On the basis of the discovery of a novel family of peptidases

与致命的神经退行性疾病相关的人类 CLN2 的生化特征：基于新型肽酶家族的发现

• 批准号: 15380072
• 负责人: ODA Kohei
• 金额: $ 8.13万
• 依托单位: Kyoto Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380072/
• 关键词: fatal neurodegenerative disease; serine-carboxyl pepetidases; CLN2; novel peptidase family; sedolisin

My colleagues and I determined the crystal structure of sedolisin at high resolution and defined a novel family, serine-carboxyl peptidases (sedolisins, MEROPS S53 family). Unique features of this family are as follows ; (1)subtilisin-like fold, (2)catalytic triad consisting of Ser, Glu, and Asp, (3)involvement of asparatate in the formation of an oxyanion hole. CLN2 plays a crucial role in lysosomal protein degradation and deficiency of this enzyme leads to a fatal neurodegenerative disease (Batten disease).In this -project, structure and function relationship of CLN2 was studied for analyzing biochemical process of Batten disease.(1)Catalytic function of h residues Ser280 and Glu77The catalytic mechanism of this family was postulated to utilize glutamate (Glu77) as a general base abstracting a proton from the serine (Ser280) that acts as a nucleophile. The S280A mutant of CLN2 showed no enzymatic activity. In addition, CLN2 was inactivated by Ac-IAF-CHO, which bind covalently to the … More catalytic serine residue. The E77A mutant did not show any significant enzymatic activity (1/10^4 lower than that of wild-type enzyme). Coupled with the results of structure analysis, the residues Ser280 and Glu77 were identified as the catalytic residues of CLN2.(2)Subsite structure of CLN2CLN2 is composed of only three subsites on the non-prime side and at least three on the prime side (S_3-S_3'). CLN2 favored Phe, Tyr, or Leu at the P_1 position, suggesting that the S_1 subsite is occupied by hydrophobic amino acid residues. CLN2 preferred Ala, Arg, or Asp at the P_2 position. The result suggests that the S_2 subsite has electrostatic interactions. CLN2 prefers Ala at the P_3 position, suggesting that the S_3 subsite is small. The results described here are well consistent with the structure model of CLN2.(3)Homology modeling of the structure of CLN2A three-dimensional model of CLN2 was built based on the homology with sedolisin. It was clarified that the carboxyl group of Asp132 would extend out into the active site cleft and act as an anchor of the N-terminal of the substrate.(4)Crystal structure of CLN2Three-dimensional structure analyses of CLN2 complexes with and without new tripeptide-based inhibitors are currently under way. Less

我和我的同事以高分辨率确定了 sedolisin 的晶体结构，并定义了一个新的家族，丝氨酸羧基肽酶（sedolisins，MEROPS S53 家族），该家族的独特特征如下：（1）枯草杆菌蛋白酶样折叠； )由Ser、Glu和Asp组成的催化三联体，(3)天冬氨酸参与氧阴离子空穴的形成。在溶酶体蛋白质降解中起着至关重要的作用，该酶的缺乏会导致致命的神经退行性疾病（Batten病）。本项目研究CLN2的结构和功能关系，以分析Batten病的生化过程。（1）催化功能h 残基 Ser280 和 Glu77 该家族的催化机制被假定为利用谷氨酸 (Glu77) 作为从丝氨酸中提取质子的通用碱基CLN2 的 S280A 突变体没有表现出任何酶活性。此外，Ac-IAF-CHO 与催化丝氨酸残基共价结合，使 CLN2 失活。显着的酶活性（比野生型酶低1/10^4），结合结构分析结果，残留物。 Ser280和Glu77被鉴定为CLN2的催化残基。(2)CLN2CLN2的亚位点结构仅由非素侧的3个亚位点和素侧的至少3个亚位点(S_3-S_3')组成。 P_1 位置的 Tyr 或 Leu，表明 S_1 亚位点被 CLN2 优先的疏水性氨基酸残基占据。 P_2 位置上的 Ala、Arg 或 Asp 表明 CLN2 更喜欢 P_3 位置上的 Ala，这表明此处描述的结果与结构模型非常一致。 (3)CLN2结构的同源建模基于与sedolisin的同源性，建立了CLN2的三维模型。 Asp132的羧基会延伸到活性位点裂缝中并充当底物N端的锚定点。(4)CLN2的晶体结构对有和没有新型三肽抑制剂的CLN2复合物进行了三维结构分析目前正在进行中。

项目成果

Wlodawer, A.: "A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases"BMC Struct.Biol.. 3・1. 8 (2003)

Wlodawer, A.：“三肽基肽酶 I (CLN2) 的模型，丝氨酸羧基肽酶 sedolisin 家族中普遍存在且高度保守的成员”BMC Struct.Biol.. 3・1 (2003)。

新規プロテアーゼファミリー・sedolisinの構造と機能

新型蛋白酶家族sedolisin的结构和功能

• 发表时间: 2003
• 期刊: バイオサイエンスとバイオインダストリー 61
• 作者: Suda K.;Udagawa N.;Sato N.;Takami M.;Itoh K.;Woo;J.T;Takahashi N.;Nagai K.;小田 耕平
• 通讯作者: 小田 耕平

1.2 Åclystal structure of the serine carboxyl proteinase pro-kumamolisin ; structure of an intact pro-subtilase.

1.2 丝氨酸羧基蛋白酶原 kumamolisin 的晶体结构；完整枯草杆菌酶原的结构。

• 发表时间: 2004
• 期刊: Structure(Camb) 12
• 作者: Suda K.;Udagawa N.;Sato N.;Takami M.;Itoh K.;Woo;J.T.;Takahashi N.;Nagai K.;Comellas-Bigler M
• 通讯作者: Comellas-Bigler M

小田 耕平: "新規プロテアーゼファミリー、セドリシンの発見"科学と工業. 78・4. 1-7 (2004)

小田晃平：“新蛋白酶家族塞多里斯的发现”《科学与工业》78・4（2004）。

小田 耕平: "新規プロテアーゼファミリーsedolisinの構造と機能"バイオサイエンスとインダストリー. 61・8. 11-16 (2003)

小田晃平：“新型蛋白酶家族sedolisin的结构和功能”生物科学与工业61・8（2003）。