Structural Analysis of the Mannose 6-Phosphate Receptors

甘露糖 6-磷酸受体的结构分析

• 批准号: 10327288
• 负责人: Nancy M. Dahms
• 金额: $ 60.95万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327288
• 关键词: Address; Alpha-galactosidase; Animal Model; Binding; Binding Sites; Biochemical; Biodistribution; Biogenesis; Biological Assay; Catabolism; Cell Death; Cell Survival; Cell physiology; Cells; Cellular Assay; Child; Complex; Crystallization; Data; Data Reporting; Differentiation and Growth; Disease; Dissociation; Electron Microscopy; Enzymes; Excision; FDA approved; Fabry Disease; Family Characteristics; Functional disorder; Genetic Diseases; Glycoside Hydrolases; Glycosphingolipids; Goals; Homeostasis; Housing; Human; Hydrolase; IGF Type 2 Receptor; IGF2R gene; Incidence; Inherited; Insulin-Like Growth Factor II; Intravenous infusion procedures; Kinetics; Knowledge; Ligand Binding; Ligands; Link; Longevity; Lysosomal Storage Diseases; Lysosomes; Maintenance; Mass Spectrum Analysis; Measures; Mediating; Mendelian disorder; Metabolism; Modeling; Molecular; Molecular Conformation; Mus; Mutagenesis; Mutation; N-terminal; NMR Spectroscopy; Nature; Neonatal Screening; Nutrient; Organelles; Organism; Pathogenicity; Patients; Peptide Hydrolases; Phagocytes; Physiological Processes; Plasma; Plasminogen; Play; Polysaccharides; Population Heterogeneity; Positioning Attribute; Process; Production; Property; Proteins; Rattus; Receptor Gene; Recombinants; Recycling; Resolution; Roentgen Rays; Role; Specificity; Structure; System; Techniques; Testing; Therapeutic; Tissues; Tumor Suppressor Proteins; Urokinase Plasminogen Activator Receptor; Variant; X-Ray Crystallography; antigen processing; autosomal recessive trait; base; biophysical analysis; biophysical techniques; body system; cell growth; cell growth regulation; cell motility; design; disease-causing mutation; enzyme replacement therapy; extracellular; human disease; improved; inhibitor; insight; lysosomal proteins; mannose 6 phosphate; milligram; novel; particle; protein aggregation; receptor; receptor binding; receptor function; treatment strategy

Lysosomes perform degradative metabolism critical to many endocytic, phagocytic, and autophagic processes. Cation-independent mannose 6-phosphate receptor (CI-MPR) plays a vital role in the biogenesis of lysosomes by delivering ~60 different newly synthesized hydrolytic enzymes with mannose 6-phosphate (M6P) on their N-glycans to lysosomes. Lysosomal storage diseases (LSDs) are caused by mutations in lysosomal proteins, mainly enzymes, that result in defective catabolism and substrate accumulation. Characteristic of the family of ~70 LSDs is their progressive and debilitating nature due to their impact on multiple organ systems. Treatment is symptomatic for most LSDs, with only 11 having FDA-approved therapies. CI-MPR's ability to internalize recombinant M6P- containing enzymes delivered to patients by bi-weekly intravenous infusion forms the basis of enzyme replacement therapy (ERT) for 9 of these therapies. However, structural knowledge of the interaction between CI-MPR and its cargo of ~60 different lysosomal enzymes is lacking. CI-MPR also binds a diverse set of extracellular non-M6P-containing ligands that mediate CI-MPR's tumor suppressor role and regulation of cell growth and differentiation. CI-MPR regulates plasma insulin-like growth factor 2 (IGF2) levels, and binds three components of the plasminogen activation system, plasminogen, urokinase-type plasminogen activator receptor (uPAR), and tissue-type PA (tPA). However, limited information is available concerning the molecular basis for CI-MPR's interaction with plasminogen and uPAR. Here we will expand upon our preliminary data that: 1) revealed the crystal structure of CI- MPR's N-terminal region that houses several ligand binding sites, 2) showed the first structural view of CI-MPR's entire extracellular region by high-resolution electron microscopy (EM), and 3) identified conformational changes elicited by pH and ligand binding. The overall structure of CI-MPR's 2300- residue extracellular region comprised of 15 domains will be determined alone and bound to ligands using an integrated approach combining single particle EM, mass spectrometry, X-ray crystallography and NMR spectroscopy. The first structure of a complex between CI-MPR and a lysosomal enzyme (Aim 1), plasminogen (Aim 2), and uPAR (Aim 2) will be elucidated. Kinetic analyses and cell-based assays will evaluate allosteric effects of each of CI-MPR's ligands (Aim 2). The mechanism of acidic pH-dependent ligand dissociation, which is essential to the functioning of CI-MPR and other endocytic receptors, will be probed using mutagenesis studies and NMR techniques (Aim 3). Our new rat model of the LSD Fabry disease will be used to test novel lysosomal enzyme-IGF2 fusion constructs for their ability to reduce substrate accumulation (Aim 4). These studies will provide insight for the design of improved therapeutics for the treatment of LSDs, and novel inhibitors of plasminogen activation.

溶酶体对许多内吞，吞噬和自噬至关重要的降解代谢 过程。阳离子非依赖性甘露糖6-磷酸受体（CI-MPR）在 通过递送约60种不同新合成的水解酶的溶酶体生物发生 在其N-聚糖上向溶酶体上的6-磷酸甘露糖（M6P）。溶酶体储存疾病（LSD） 是由溶酶体蛋白（主要是酶的突变）引起的，导致分解代谢缺陷 和底物积累。 〜70 LSD家族的特征是他们的进步和 由于其对多器官系统的影响而使性质使人衰弱。对于大多数治疗是有症状的 LSD，只有11种具有FDA批准的疗法。 CI-MPR能够内在化重组M6P- 每两周静脉输液递送给患者的酶是酶的基础 这些疗法中的9种替代疗法（ERT）。但是，互动的结构知识 在CI-MPR及其约60种不同溶酶体酶之间的货物之间缺乏。 CI-MPR也绑定 介导CI-MPR的肿瘤抑制作用的各种细胞外非M6P的配体 和细胞生长和分化的调节。 CI-MPR调节血浆胰岛素样生长因子2 （IGF2）水平，并结合纤溶酶原激活系统纤溶酶原的三个成分 尿激酶型纤溶酶原活化剂受体（UPAR）和组织型PA（TPA）。但是，有限 有关CI-MPR与纤溶酶原和CI-MPR相互作用的分子基础的信息 UPAR。在这里，我们将扩展我们的初步数据：1）揭示了CI-的晶体结构 MPR的N末端区域容纳多个配体结合位点，2）显示了第一个结构视图 通过高分辨率电子显微镜（EM）和3）确定的CI-MPR的整个细胞外区域 pH和配体结合引起的构象变化。 CI-MPR的2300-的整体结构 由15个结构域组成的残留细胞外区域将单独确定并与配体结合 使用结合单个粒子EM，质谱，X射线晶体学的集成方法 和NMR光谱。 CI-MPR和溶酶体酶之间复合物的第一个结构 （AIM 1），纤溶酶原（AIM 2）和UPAR（AIM 2）将被阐明。动力学分析和基于细胞的分析 测定将评估CI-MPR的每个配体的变构作用（AIM 2）。酸性的机制 pH依赖性配体解离，这对于CI-MPR和其他内吞作用至关重要 受体将使用诱变研究和NMR技术进行探测（AIM 3）。我们的新老鼠模型 LSD Fabry病将用于测试新型的溶酶体酶-IGF2融合构建体 减少底物积累的能力（目标4）。这些研究将为设计提供见解 改善了治疗LSD的治疗疗法，以及纤溶酶原活化的新型抑制剂。