PATHOGENESIS OF LYSOSOMAL ACID LIPASE DEFICIENCY

溶酶体酸性脂肪酶缺乏症的发病机制

• 批准号: 2740966
• 负责人: HONG DU
• 金额: $ 14.93万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2740966
• 关键词: Wolman's disease; cholesterol ester storage disease; disease /disorder etiology; enzyme activity; enzyme deficiency; enzyme mechanism; enzyme substrate; esterase inhibitor; gene targeting; genetic models; genetically modified animals; laboratory mouse; model design /development; phenotype; point mutation; site directed mutagenesis; sterol esterase

The objectives of the proposed studies are to elucidate the pathogenesis of Wolman (WD) and Cholesteryl Ester Storage (CESD) diseases. These diseases are due to the mutations at the lal locus that leads to a deficiency of lysosomal acid lipase (LAL). This enzyme has activity toward triglyceride and cholesteryl ester substrates and the determinates of the substrate specificity, and, therefore, the phenotypes is not understood. Using our human and mouse LAL cDNAs and pET 21A and baculovirus expression systems, we have produced sufficient purified LAL for making specific antibody, and the initial purification and characterization of normal and mutagenized LAL. We have cloned the mLAL gene and created a "knock-out" mouse at this locus. Using this animal model and our in vitro heterologous expression system, we propose to: 1) Characterize the phenotype of the lal-/lal-mouse by natural history, histologic and lipid characterization approaches. Based on the rat model anticipate that our liveborn (survival for 21 days at least) lal-/lal- mice will have a WD phenotype. 2) Normal and selected hLAL and mLAL mutant forms will be expressed characterized by detailed kinetic analyses to determine residues important for the substrate preference, and their relationship to WD and/or CESD phenotypes. These analyses will include steady-state and transient kinetics with active site directed inhibitors, and selected substrates with differing acyl chain composition. 3) The essential N-glycosylation occupancy for catalytically active conformers will be assessed by site-directed mutagenesis of the conserved consensus sequences between hLAL, catalytically active conformers will be assessed by site-directed mutagenesis of the conserved consensus sequences between hLAL, mLAL and rLAL. 4) Based on the in vitro findings, selected specific mutations will be introduced into the lal-/lal- mice by the "knock-in" approach to determined their physiologic relevance and relationship to the WD and CESD phenotypes. The development of a fleet of mice homozygous for selected point mutations at the lal locus will provide essential reagents for a more complete delineation of the developmental progressively, tissue specific involvement, and the potential for differential tissue expression of LAL as a basis for the pathogenesis of the phenotypes. These studies will also provide a basis for future studies of enzyme and gene therapeutic approaches to WD and CESD as well as other inborn errors of metabolism.

拟议的研究的目标是阐明沃尔曼（WD）和胆汁胆固醇酯（CESD）疾病的发病机理。这些疾病是由于LAL基因座的突变导致溶酶体酸脂肪酶（LAL）缺乏。该酶对甘油三酸酯和胆固醇酯的底物具有活性，以及​​底物特异性的确定性，因此无法理解表型。使用我们的人类和小鼠LAL cDNA以及PET 21A和杆状病毒表达系统，我们制作了足够的纯化LAL来制造特定抗体，以及正常和诱变的LAL的初始纯化和表征。我们已经克隆了MLAL基因，并在此基因座创建了“淘汰”小鼠。使用这种动物模型和我们的体外异源表达系统，我们建议：1）根据自然史，组织学和脂质表征方法来表征LAL-/LAL-MOUSE的表型。基于大鼠模型，预计我们的活体发生（至少21天生存）lal-/lal-小鼠将具有WD表型。 2）正常和选定的HLAL和MLAL突变体形式将以详细的动力学分析来表达，以确定对底物偏好重要的残基及其与WD和/或CESD表型的关系。这些分析将包括具有活性位点的定向抑制剂的稳态和瞬态动力学，以及具有不同酰基链组成的底物。 3）催化活性构象体的必不可少的N-糖基化占用率将通过位于HLAL，HLAL，催化活性构象体之间的保守共识序列的位置定向诱变来评估，将通过位置定向的HLAL，MLAL，MLAL和RLAL之间的位置定向诱变进行评估。 4）基于体外发现，将通过“敲入”方法将选定的特定突变引入LAL-/LAL-小鼠中，以确定其生理相关性以及与WD和CESD表型的关系。在LAL基因座的选定点突变纯合的一组小鼠的发展将为更完整的发育，逐渐逐步描述，组织特异性参与以及LAL作为差异组织表达作为表型的发病机理的基础的潜力。这些研究还将为WD和CESD的酶和基因治疗方法以及其他天生的代谢错误提供基础。