GlcNAc-1-phosphotransferase: Structure and Lysosomal Hydrolase Recognition

GlcNAc-1-磷酸转移酶：结构和溶酶体水解酶识别

• 批准号: RGPIN-2019-06819
• 负责人: Rini, James
• 金额: $ 3.06万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716229
• 关键词: GlcNAc; phosphotransferase; Structure; Lysosomal; Hydrolase

A) Rationale/Aim: The Golgi-resident enzyme, UDP-N-acetylglucosamine-1-phosphotransferase (GNPT) initiates the addition of the mannose-6-phosphate (M6P) moiety to the N-glycans of soluble lysosomal hydrolases. This modification is required for the targeting of these enzyme to lysosomes but how GNPT recognizes its structurally diverse substrates is unknown. The short-term goal of this work (the subject of this proposal) is to determine the structure of GNPT and insight into the basis for its substrate specificity. In the long-term we seek to fully describe the basis for GNPT's ability to specifically recognize its over 50 lysosomal enzyme substrates. B) Background: GNPT is a ~360 kDa complex composed of three subunits (222). Each of these subunits contain multiple domains that play different roles in binding and catalysis. Among these, the so-called stealth domain (a sugar-phosphate transferase domain in some bacteria) forms the catalytic core of GNPT and the N1, N2 and DMAP protein interaction domains are involved in substrate recognition. Notably, the subunit is not required for the recognition of some lysosomal enzymes and the protein interaction domains play different roles in the recognition of different substrates. C) Preliminary Results: We have expressed the human GNPTAB (the and subunits are generated by proteolysis) and GNPTG cDNA's both separately and in combination using our PiggyBac-based mammalian cell expression system. Various forms of the enzyme have been characterized by negative strain EM and our preliminary cryo-EM analysis of one of them establishes that it can be successfully frozen in thin ice. The resulting 2D class averages from the cryo data provide evidence of 2-fold symmetry and a sample suitable for high-resolution structure determination. We have also expressed several lysosomal enzymes to be used in characterizing the specificity of GNPT. D) Approach: Single particle cryo-EM analysis will be used to determine the structure of various forms of GNPT. In addition, x-ray crystal structures of GNPT domains/fragments will be determined to add high resolution details. Key among the x-ray structures will be an active form of GNPT lacking the protein interaction domains and the entire subunit and two homologous bacterial stealth domains. In addition, biochemical analysis will be used to determine the role played by various GNPT domains/subunits in the recognition of selected lysosomal enzyme substrates. E) Outcomes: This work will lead to the first structure of GNPT and/or fragments of it including the catalytic core or stealth domain conserved across evolution. GNPT uses different domains to bind different lysosomal enzymes and the work will also provide insight into the structural and biochemical basis for that process.

A）理由/目的：高尔基体驻留酶UDP-N-乙酰葡糖胺-1-磷酸转移酶（GNPT）启动甘露糖-6-磷酸（M6 P）部分添加到可溶性溶酶体水解酶的N-聚糖中。 这种修饰是这些酶靶向溶酶体所必需的，但GNPT如何识别其结构多样的底物尚不清楚。 这项工作的短期目标（本提案的主题）是确定GNPT的结构，并深入了解其底物特异性的基础。 从长远来看，我们试图充分描述GNPT特异性识别其超过50种溶酶体酶底物的能力的基础。 B）背景：GNPT是由三个亚基组成的~360 kDa复合物（222）。 这些亚基中的每一个都包含在结合和催化中发挥不同作用的多个结构域。 其中，所谓的隐形结构域（在某些细菌中的糖-磷酸转移酶结构域）形成GNPT的催化核心，而N1、N2和DMAP蛋白相互作用结构域参与底物识别。 值得注意的是，亚基是不需要的一些溶酶体酶的识别和蛋白质相互作用结构域在不同的底物的识别中发挥不同的作用。 （一）初步结果：我们已经表达了人GNPTAB（和亚基通过蛋白水解产生）和GNPTG cDNA的单独和组合使用我们的PiggyBac为基础的哺乳动物细胞表达系统。 阴性菌株EM已对该酶的各种形式进行了表征，我们对其中一种酶的初步冷冻EM分析表明，它可以成功地冷冻在薄冰中。 从低温数据得到的2D类平均值提供了2倍对称性的证据和适合于高分辨率结构测定的样品。 我们还表达了几种用于表征GNPT特异性的溶酶体酶。 D）方法：将使用单颗粒冷冻-EM分析来确定GNPT的各种形式的结构。 此外，将确定GNPT结构域/片段的X射线晶体结构以增加高分辨率细节。X射线结构中的关键将是GNPT的活性形式，缺乏蛋白质相互作用结构域和整个亚基以及两个同源细菌隐形结构域。 此外，生化分析将用于确定各种GNPT结构域/亚基在识别选定的溶酶体酶底物中所起的作用。 E）结果：这项工作将导致GNPT的第一个结构和/或它的片段，包括在进化过程中保守的催化核心或隐形结构域。 GNPT使用不同的结构域来结合不同的溶酶体酶，这项工作也将为该过程的结构和生化基础提供深入了解。