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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688480
• 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)原理/目的：高尔基体驻留酶，UDP-N-乙酰氨基葡萄糖-1-磷酸转移酶(GNPT)启动甘露糖-6-磷酸(M6P)部分加到可溶性溶酶体水解酶的N-糖链上。这种修饰是将这些酶定向到溶酶体所必需的，但GNPT如何识别其结构多样化的底物尚不清楚。这项工作(本提案的主题)的短期目标是确定GNPT的结构，并深入了解其底物特异性的基础。从长远来看，我们寻求全面描述GNPT特定识别其50多种溶酶体酶底物的能力的基础。背景：GNPT是由3个亚基(222)组成的约360 kDa的复合体。每个亚基都包含多个结构域，在结合和催化中发挥不同的作用。其中，所谓的隐形结构域(在一些细菌中是糖-磷酸转移酶结构域)构成了GNPT的催化核心，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，一些溶酶体酶的识别并不需要亚基，蛋白质相互作用结构域在识别不同底物方面发挥着不同的作用。*C)初步结果：利用我们的基于piggyBac的哺乳动物细胞表达系统，我们分别和联合表达了人GNPTAB(和的亚基是由蛋白质降解产生的)和GNPTG的cDNAs。各种形式的酶已经用负应变EM进行了表征，我们对其中一种酶的初步冷冻-EM分析表明，它可以成功地在薄冰中冷冻。从低温数据得到的2D类平均值提供了2重对称性的证据，并提供了一个适合高分辨率结构确定的样本。我们还表达了几种溶酶体酶，用于表征GNPT的特异性。*D)方法：将使用单颗粒冷冻-EM分析来确定各种形式的GNPT的结构。此外，还将确定GNPT结构域/碎片的X射线晶体结构，以增加高分辨率细节。X射线结构中的关键是一种活性形式的GNPT，它缺乏蛋白质相互作用结构域和整个亚单位以及两个同源的细菌隐形结构域。此外，生化分析将被用来确定不同的GNPT结构域/亚单位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/或其片段，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，这项工作也将为这一过程的结构和生化基础提供洞察力。