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

我和我的同事在高分辨率下测定了七叶皂素的晶体结构，并定义了一个新的家族，丝氨酸-羧基肽酶(sedolisins，MeropsS53家族)。该家族的特点如下：(1)枯草杆菌素样折叠；(2)由丝氨酸、谷氨酸和天冬氨酸组成的催化三联体；(3)天冬氨酸参与氧阴离子空穴的形成。CLN2在溶酶体蛋白降解过程中起着至关重要的作用，该酶的缺失会导致一种致命性的神经退行性疾病(巴登病)。本项目研究了CLN2的结构和功能关系，以分析巴顿病的生化过程：(1)h残基Ser280和Glu77的催化功能。推测该家族的催化机制是利用谷氨酸(Glu77)作为一般碱从丝氨酸(Ser280)中提取质子作为亲核剂。CLN2的S280A突变体没有显示出酶活性。此外，通过与…共价结合的Ac-iaf-CHO使CLn2失活更多催化丝氨酸残基。E77A突变体没有表现出任何显著的酶活性(比野生型酶低1/104)。结合结构分析结果，确定Ser280和Glu77残基为CLN_2的催化残基。(2)CLN_2CLN_2的亚位结构由非素侧的3个亚位和素侧的至少3个亚位(S_3-S_3‘)组成。CLN2Phe、Tyr或Leu位于P_1位，提示S_1亚基被疏水氨基酸残基占据。在P_2位，CLN_2偏爱Ala、Arg或Asp。结果表明，S_2亚位存在静电相互作用。Cln2偏爱P3位Ala，提示S 3亚位较小。所得结果与CLN_2的结构模型吻合较好。(3)建立了CLN_2的结构同源模型，建立了CLN_2的三维结构模型。明确了Asp132的羧基将延伸到活性中心缝隙中，并作为底物N-末端的锚。(4)CLN_2的晶体结构目前正在对CLN_2络合物进行三维结构分析。较少

项目成果

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

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

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)。

Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate.

两个抑制剂分子结合在假单胞菌 sedolisin 的活性位点：肽底物裂解后的副产物复合物模型。

• DOI: 10.1016/j.bbrc.2003.12.130
• 发表时间: 2004
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Wlodawer,Alexander;Li,Mi;Gustchina,Alla;Oyama,Hiroshi;Oda,Kohei;Beyer,BretB;Clemente,Jose;Dunn,BenM
• 通讯作者: Dunn,BenM

Wlodawer, A.: "Structural and enzymatic properties of the sedolisin family of serine-carboxyl peptidases"Acta Biochim.Pol.. 50・1. 81-102 (2003)

Wlodawer, A.：“丝氨酸羧基肽酶的 sedolisin 家族的结构和酶学特性”Acta Biochim.Pol.. 50・1 (2003)。